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Two cars, a B-double and two smaller trucks were all involved in the crash on the Western Highway, near the intersection of Thomas Road in Armstrong, about 12.30pm, according to police.
The driver of one of the cars and two occupants of one of the trucks were all pronounced dead at the scene.
Two people have been taken to hospital, including the driver of the second car.
Police remain on scene, where the highway is closed in both directions between Ararat and Great Western.
Motorists are advised to detour through Moyston using Ararat-Halls Gap Road and Moyston-Great Western Road.
The detour will add an additional 20 kilometres to motorists’ journey, so drivers are urged to allow for plenty of extra time.
The highway is expected to be closed for most of the day.
The exact circumstances of the collision are yet to be determined and investigations remain ongoing.