The Ontario Provincial Police issued a news release on Wednesday identifying the victims as 38-year-old Anand Vora and 37-year-old Jasjit Sandhu.
Police say Vora was the driver.
The deadly crash occurred late Tuesday morning when the tractor-trailer went off the eastbound lanes of the highway, west of the Trussler Road overpass near Cambridge, Ont., and ended up in the Nith River.
Police recovered the bodies of the two victims from the scene.
The eastbound lanes of the highway remained closed for 11 hours after the crash.
The OPP say that partial lane closures will occur at times on Wednesday as officers continue to investigate the crash.