From south to north, the route passes through the cities of Jackson Center, Uniopolis, Lima, Columbus Grove, Ottawa, Leipsic, Belmore, McClure, Grand Rapids, Perrysburg, Rossford, and Toledo.

Route 65 parallels the south bank of the Maumee River for its northernmost 32 miles, from east of Napoleon to Toledo.

Its original southern terminus was at State Route 15 in Ottawa, and it crossed the Maumee River near Liberty Center, continuing northward to Delta and terminating at the Michigan state line.

In June, 2007, the Veterans' Glass City Skyway opened, taking Interstate 280 across the Maumee.

Subsequently, State Route 65 was rerouted onto the Robert Craig Memorial Bridge, the former crossing of Interstate 280 across the river, the week after the new bridge opened,[2] ending at the former Front Street interchange.