Located in the Eastern Panhandle, Jefferson County is the easternmost county of West Virginia, bounded by the Potomac River and Maryland on the north; the Blue Ridge Mountains and Loudoun County, Virginia, on the east; Clarke County, Virginia, on the south; and Berkeley County on the west. It occupies 211.6 square miles. Its municipalities include Charles Town, the county seat (established in 1786 by George Washington's brother Charles Washington); Ranson (1910); Harpers Ferry (1851); Bolivar (1825); and Shepherdstown (1762). Jefferson County was established from a portion of Berkeley County by the Virginia General Assembly on October 26, 1801. It was named for Thomas Jefferson.

The earliest permanent European-American settlement probably occurred in the second decade of the 18th century. Land speculators Joist Hite and John and Abraham Van Meter acquired large tracts in the Northern Neck of Virginia, which included present Jefferson County. Land was sold to German and English immigrants, many of them arriving from Pennsylvania, New Jersey, and Delaware. Shortly thereafter, other settlers arrived from the Tidewater area of Virginia. Many of the later arrivals received land from Lord Thomas Fairfax. As Hite's settlers and those holding grants from Fairfax began to lay claim to the same property, Fairfax and Hite engaged in a lawsuit in 1749. The suit was not settled until 1786, leaving local land titles in dispute for many years.

During the 1740s and 1750s, George Washington surveyed several plats in the Jefferson County area for Fairfax. Using his surveying fees, he purchased his first piece of land, from Robert Rutherford on Bullskin Run. Washington's brothers also acquired land in present Jefferson County, and the family flourished there until the Civil War. More Washington family descendants are buried in Jefferson County than in any other place. Several homes built by family members still grace the countryside.

The arrival from the north and east by Germans, English, and Scotch-Irish, and from the south of Virginia planters, created a cultural mix that persisted for much of the county's history. Shepherdstown and Harpers Ferry looked northward, developing strong economic ties with Baltimore and Hagerstown, Maryland, and Philadelphia, as well as nearby Martinsburg. Charles Town and Smithfield maintained ties with Winchester and the South. During the 1700s, the enslaved population was not large and was concentrated mostly on a few plantations, but by 1850, enslaved people numbered 3,960, or 27 percent of the total population.

For most of its history, Jefferson County's economy has been primarily agricultural, producing grain and other crops in the 18th and 19th centuries and orchard fruit in the late 19th and early 20th centuries. Limestone quarrying has also been important. Deposits of iron ore spurred the development of an iron industry along the Shenandoah River as early as 1742, and at Friend's Orebank along the Potomac east of Bakerton in the 1760s. Friend's Orebank operated for a century and a half, until World War I.

Two important events that shaped the future of Jefferson County were the establishment of the federal armory at Harpers Ferry in 1799 and the arrival of the Baltimore & Ohio Railroad (B&O) in 1834. John Brown's raid on Harpers Ferry was prompted by the presence of the armory, the town's proximity to defensible positions in the mountains, and the belief that the surrounding enslaved population would rise up and support the insurgents. Martin Robison Delany, born in Charles Town, was among 34 blacks who had met with Brown in Canada prior to the 1859 raid to develop a provisional constitution for the liberated slaves. During the Civil War, Delany became the first Black field officer in the U.S. Army.

The presence of the B&O influenced the decision in 1863 to include Jefferson County in the new state of West Virginia, although most residents had sided with Virginia when the mother state seceded from the Union. Disenfranchised after the war because of their support of the Confederacy, they were unable to prevent the transfer of the courthouse from Charles Town to Shepherdstown in 1865. The county seat was returned to Charles Town in 1871, and relations between the two towns remained cool for decades.

Two treason trials were held in the Jefferson County courthouse. The first trial was of John Brown and the raiders who survived the 1859 raid. Brown was found guilty and hanged. A second series of treason trials was held at the same site in 1922. Defendants included Bill Blizzard, who allegedly led coal miners in the armed march culminating in the Battle of Blair Mountain. No one was convicted of treason in 1922. In 2023, the Jefferson County Courthouse was designated as a National Historic Landmark, noting the significance of both the Brown and Mine Wars treason trials.

Jefferson County's economy, particularly its growing industrial base, was hurt by the Civil War. The Harpers Ferry armory was not reopened after the war, and part of its facilities were used to establish an educational institution for African Americans, Storer College, in 1867. Shepherd Normal School, established in Shepherdstown in 1871, later became Shepherd University.

Jefferson County's population grew slowly from the end of the Civil War until World War II, to nearly 16,000. Agriculture remained the major source of revenue, with corn and wheat being the major crops. The orchard industry was introduced in the 1880s, and many dairy farms were established in the 1920s and 1930s. During the 1980s and 1990s, the number of farms declined, partly because of the decreased profitability of farming and partly because of the pressure exerted by residential development. By the year 2000, raising beef cattle and horse breeding were the most promising areas for agricultural growth. With the opening of the Charles Town Racetrack (now Hollywood Casino at Charles Town Races) in 1933, Jefferson County became the first track in the state to offer parimutuel betting. The racetrack is still one of the major employers in the area.

In 1944, Congressman Jennings Randolph introduced legislation authorizing the purchase of land in Harpers Ferry for establishment of a national park. Harpers Ferry National Historical Park grew out of this effort. The facilities of Storer College were absorbed into the park when the college closed in 1960. The National Park Service also maintains design and interpretive centers at Harpers Ferry, which serve Park Service units nationwide. Since 1997, the U.S. Fish and Wildlife Service has operated its National Conservation Training Center at Shepherdstown. Recreational and historic tourism account for an increasing number of visitors to Jefferson County as well as growing revenues.

Like much of the neighboring area, Jefferson County began to grow more rapidly in the 1970s. Between 1970 and 2020, the number of residents increased from 21,280 to 57,701. From just 2000 to 2020, the population grew by 37 percent, making it one of the fastest-growing counties in the state. During this period, the county experienced much residential growth without a proportionate increase in its business and industrial base, as the majority of its people commuted outside the county to work. In 2023, Jefferson County's largest employers were the county school system, the casino and racetrack, Shepherd University, WVU Medicine, and American Public University System

The county is part of the metropolitan Washington area. In the 21st century, Jefferson County faces the challenge of balancing population growth with the need to preserve the environmental and historic resources that attract people to the area.