When the highway was originally established in 1926, the route extended from North Augusta, South Carolina, to Port Huron, Michigan.

It then stays at a general north route through the cities of Statesboro, Millen, Waynesboro, and finally Augusta, where it crosses the Savannah River into South Carolina.

Splitting from I-26/US 74 near Fletcher, it goes north through Arden, Biltmore Forest, and downtown Asheville, before reconnecting with I-26 near Woodfin.

Along the northern bank of the French Broad River, it crosses back over and leaves the Cherokee National Forest.

US 25 enters Newport, after crossing the Pigeon River, and serves as Broadway Street through the downtown area.

Traversing a 145.7 miles (234.5 km) from Newport, Tennessee, to North Corbin, Kentucky, US 25W goes west connecting Dandridge and Knoxville and northwest to Clinton.

In 1974, US 25 was eliminated in Ohio and Michigan, establishing its northern terminus on the Clay Wade Bailey Bridge in Covington, Kentucky.

In 2000, US 25E was rerouted through the Cumberland Gap Tunnel (opened four years prior), making a direct link between Tennessee and Kentucky, eliminating Virginia's short section.