During the Protohistoric period (1550-1690 A.D.), Indian villages in present-day West Virginia had access to European trade goods but no direct contact with Europeans. The Fort Ancient sites include the Buffalo, Clover, and Rolfe sites, with Rolfe being the latest, dating to 1640. Other Fort Ancient sites, such as Orchard, Marmet, Logan, and Snidow, also date to this time, but many have been destroyed. A key artifact found at these sites is the shell gorget, which shows connections to villages in eastern Tennessee.

In the Northern Panhandle, the Protohistoric Monongahela sites include LaPoe and Hughes Farm. On the South Branch of the Potomac, the Iroquois-speaking Susquehannock occupied a series of villages from 1590 to the 1630s A.D.. Prominent Susquehannock villages were located at Moorefield and Herriott Farm.

The descendants of many protohistoric villagers remain unknown. By the mid-1675, the villages had been abandoned due to attacks from the Iroquois. By the 1700s, the Shawnee, Cherokee, and Iroquois used West Virginia as a hunting ground. The Shawnee may have descended from multicultural villages, such as Orchard, but burial practices suggest that most of the West Virginia Fort Ancient villages were connected to Siouan-speaking groups from Virginia, the Carolinas, and the Mississippi Valley.