The Northwestern Virginia Turnpike was chartered by the General Assembly of Virginia in 1827, to connect Winchester to Parkersburg. Later known as U.S. 50, it soon became the most important east-west road in the state. Planned as the major rival to the National Road, this route linked a significant portion of northwestern Virginia to Baltimore, via Winchester, rather than to Richmond and the Tidewater, contributing to Western Virginia's Unionist tendencies in the Civil War. The Northwestern Turnpike, which ran generally parallel to the later Baltimore & Ohio and the Northwestern Virginia railroads, declined in importance from the mid-1850s.

From the beginning, the Northwestern Turnpike and the Staunton-Parkersburg Turnpike were rivals in a race to the Ohio River. Winchester and Staunton both pressured the General Assembly for favorable treatment of "their" routes. But the Northwestern Turnpike was better organized in that it was placed at the outset under control of a state board of directors including the governor. The last section was finished to Parkersburg in 1838, and all bridges were completed by 1840, a decade before the Staunton road was in continuous operation.

Col. Claudius Crozet, Virginia's chief engineer, surveyed the route from Winchester through Hampshire County by way of Capon Bridge to Romney and across the South Branch in Mineral County to Patterson's Creek. Then he proposed that the road cross eight miles of Maryland and reenter Virginia at Aurora in Preston County. Though he was criticized in Virginia for a plan that would traverse another state, Crozet maintained the viability of the survey, insisting that it would allow for a branch connecting to the National Road, perhaps diverting some traffic to Winchester. From Aurora, the turnpike crossed the Cheat River, then passed through Rowlesburg, Grafton, Pruntytown, and Bridgeport to Clarksburg on the West Fork River.

Crozet's successor, Charles B. Shaw, favored Parkersburg over Sistersville as the terminus on the Ohio River, since it was in a direct line to Cincinnati. Under his direction, the route continued from Clarksburg to Salem, West Union, Pennsboro, Ellenboro, and Murphytown to Parkersburg, completing the last mileage in 1838. As was the case with all the early turnpikes, the directors and engineers faced continuing problems with labor shortages, contractor incompetence, and right-of-way problems. Since the project was financed by the state, lack of funding was not a major drawback, as it was with the Staunton-Parkersburg Turnpike.

The Northwestern Turnpike, passing as it did through the more populous counties of Western Virginia, was immediately successful. Toll collections were adequate, and profits permitted the early macadamizing of much of the route. As early as 1841, Nathaniel Kuykendall, former superintendent of the eastern section, operated a stagecoach and mail delivery service. Nonetheless, problems typical of 19th-century roads plagued the turnpike. For example, the damage caused by rains and spring flooding required constant repair. In spite of increasing competition from the railroad, the turnpike operated at a small profit and was able to meet its financial obligations throughout the 1850s. The Civil War was unkind to the Northwestern Turnpike. The new state of West Virginia was slow to assume responsibility, and by the end of the war the road was nearly impassible. Inadequately maintained in the late 19th century, it finally became part of the U.S. highway system in the 20th century.