Police are confident the scene has been cleared of all passengers and staff aboard the train.
However, they are still working with the train company to confirm passenger numbers.
The train was travelling from Sydney to Melbourne when several carriages and the engine derailed near the Hume Fwy just before 8pm.
The driver, a 54-year-old ACT man, and the train pilot, a 49-year-old Castlemaine woman, died at the scene.
Twelve passengers were transported to hospital with non-life threatening injuries.
A report for the coroner will be prepared.
Police will investigate the cause of the incident in conjunction with National Rail Safety Regulator, Australian Transport Safety Bureau, and WorkSafe.
Anyone who was on the train and hasn’t spoken with emergency services or who purchased a ticket but did not board the train is asked to call Seymour Police Station on 5735 0200.