All four people inside the sedan died at the scene.
The driver of the truck, a 30-year-old man, was rushed to Wodonga Hospital, with non-life-threatening injuries.
Chiltern Valley is located in between Wodonga and Yarrawonga, about 292km from Melbourne.
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Major Collision Investigation Unit detectives will travel to the scene, as police investigate the circumstances surrounding the crash.
Northbound traffic on the Hume Highway is closed from Springhurst Road.
