The 5.8-kilometre (3.6 mi) highway travels across the Quinte Skyway and through the Tyendinaga Mohawk Territory south of Marysville.

A previous iteration of Highway 49 existed between 1936 and 1961 from Kleinburg west to the York County boundary south of Bolton, which is today known as York Regional Road 49 (Nashville Road).

The route remained unchanged until the late 1990s, when more than half of the highway was transferred to the jurisdiction of local governments.

However, the Quinte Skyway (constructed in 1967)[6] and the section lying within the Tyendinaga Mohawk Territory remain under provincial jurisdiction.

[9] The route remained as-is for 25 years before being transferred back to York County at some point in 1961.

[13][16] Construction proceeded simultaneously on realigning the highway south to Picton, bypassing several portions and building a new road north from Roblin Mills in the process.

As part of a series of budget cuts initiated by premier Mike Harris under his Common Sense Revolution platform in 1995, numerous highways deemed to no longer be of significance to the provincial network were decommissioned and responsibility for the routes transferred to a lower level of government, a process referred to as downloading.

[1] The following table lists the major junctions along Highway 49, as noted by the Ministry of Transportation of Ontario.