The collision happened on the southbound carriageway of the M90 near Dunfermline at around 3pm yesterday.
It involved a black BMW X3 and a blue Dacia Duster car with both vehciles landing upside down.
A 69-year-old woman suffered a broken ankle and a 61-year-old is being treated for a fractured sternum at the Victoria Hospital in Kirkcaldy, police said.
Officers are appealing for any witnesses to the crash to contact them and particularly want to speak to the driver of a car that passed the accident.
A force spokesman said: "We are keen to trace the driver of a silver coloured 4x4 vehicle, which was also driving south at the time of the collision and may have witnessed the event.
"Any other person who may have witnessed the collision are urged to contact Police Scotland, Divisional Road Policing Unit, Glenrothes on 101."