It runs for 165.45 miles (266.27 km) from US 64 south of Clifton, Tennessee to an intersection with US 60 in Marion, Kentucky.

[9] U.S. Route 641 was designated by AASHO in 1951 out of a desire on the part of Indiana, Kentucky and Tennessee highway officials to create a single numbered route to connect Memphis, Tennessee to Evansville, Indiana via the popular tourist attraction Kentucky Dam.

Indiana highway officials, in their request to decommission the route, advised that 641 "serves no useful purpose" and had, indeed, not been marked in Evansville for "a number of years."

The route makes a sharp left turn at Chestnut Street and heads west before finally terminating at US 641.

It begins at a junction with US 641 south of Benton, and ends at the Exit 41 interchange with Interstate 69 (formerly the Julian M. Carroll Purchase Parkway).