The first people to form substantial settlements in what is now Knoxville were indigenous people who arrived during the Woodland period (c. 1000B.C. to A.D.1000). One of the oldest artificial structures in Knoxville is a burial mound constructed during the early Mississippian culture period (c. A.D.1000–1400). The earthwork mound has been preserved, but the campus of the University of Tennessee developed around it.
Other prehistoric sites include an Early Woodland habitation area at the confluence of the Tennessee River and Knob Creek (near the Knox-Blount county line), and Dallas Phase Mississippian villages at Post Oak Island (also along the river near the Knox-Blount line), and at Bussell Island (at the mouth of the Little Tennessee River near Lenoir City).
By the 18th century, the Cherokee, an Iroquoian language people, had become the dominant tribe in the East Tennessee region; they are believed to have migrated centuries before from the Great Lakes area. They were consistently at war with the Creek (who spoke Muskogee) and Shawnee (who spoke Central Algonquian). The Cherokee people called the Knoxville area kuwanda'talun'yi, which means "Mulberry Place". Most Cherokee habitation in the area was concentrated in what the American colonists called the Overhill settlements along the Little Tennessee River, southwest of Knoxville.
The first white traders and explorers were recorded as arriving in the Tennessee Valley in the late 17th century. There is significant evidence that Spanish explorer Hernando de Soto visited Bussell Island in 1540. The first major recorded Euro-American presence in the Knoxville area was the Timberlake Expedition, which passed through the confluence of the Holston and French Broad into the Tennessee River in December 1761. Henry Timberlake, an Anglo-American emissary from the Thirteen Colonies to the Overhill settlements along the Little Tennessee River, recalled being pleased by the deep waters of the Tennessee after his party had struggled down the relatively shallow Holston for several weeks.