In the state of Minnesota, US 65 travels for 15 miles from the Iowa border to a partial interchange with Interstate 35 in Albert Lea.

Parallel to a Union Pacific rail line through farmland, the route travels northwest and passes east of Gordonsville and west of downtown Glenville towards its first interchange with I-35.

[3][4][5] At the interchange, the road enters the city of Albert Lea, expands to a four-lane divided highway and a business loop of I-35 begins, a route co-signed along US 65.

At a junction with MN 13, the route turns onto a four-lane divided highway known as Main Street and begins traveling east towards I-35.

[14] The original route of US 65 from the Iowa state line to Saint Paul had previously been established as a portion of the Jefferson Highway, an auto trail which ran from New Orleans to Winnipeg.

[9][15] When US 65 was rerouted to end in Minneapolis in 1935, the Minnesota Legislature authorized Trunk Highway 165, which provided a direct connection from Faribault to Lakeville, bypassing the cities of Dundas, Northfield, and Farmington.