The route begins at Highway 66 and travels 123.3 kilometres (76.6 mi) southeast to the Quebec border.

It initially connected Matachewan with Highway 11 at New Liskeard, but was extended east to the Quebec border in 1956.

Rue Ontario ends at Route 101 in Notre-Dame-du-Nord, 2.6 kilometres (1.6 mi) east of the provincial border.

[2] Highway 65 was first established on August 7, 1937, when the New Liskeard to Matachewan Road was assumed by the DHO in the days following its merger with the DND on April 1.

[3] The following table lists the major junctions along Highway 65, as noted by the Ministry of Transportation of Ontario.