The Hume Freeway re-opened about 1am Friday, September 1, between Springhurst and Chiltern following a collision the previous morning.
The Department of Transport and Planning thanked drivers for their patience during the closure.
Police believe a car was trying to enter the Hume Highway when it was hit by a truck, with specialist detectives heading to Chiltern on Thursday afternoon to determine the exact cause.
Wodonga Inspector Joel Hughes said early indications were that a car had turned left at the intersection of Wenkes Road and the Hume Highway, shortly before 10.30am.
The northbound truck and the car collided near the intersection.
The passenger car appeared to roll in the collision, killing four adults, while the 30-year-old truck driver sustained non-life threatening injuries.
The Ron Finemore employee has been taken to hospital.
Inspector Joel Hughes described the incident as tragic for all involved.
"It's terrible," he said.
"Unfortunately in regional Victoria this year we've seen a number of lives lost on our roads, and this is another tragic incident.
"Victoria Police are still investigating the cause of the collision, but we just want all road users to be safe on our roads.
"It's obviously terrible news for the families of these people involved, for people and emergency services that attend at these sorts of scenes."
Police are working to identify those who have lost their lives.
Other road users stopped to assist at the scene immediately after the crash.
Acting Inspector Hughes said Rutherglen police were amongst the first on scene but other officers from Wodonga, Wangaratta and surrounds have attended.
He said crashes involving four deaths were rare.
"They don't happen very often," he said.
"One life lost is far too many on our roads, but four is obviously exponentially terrible for those people involved."
Four people have died after a car and truck collided on the Hume Freeway at Chiltern.
It is understood the car and B-double truck crashed on the Hume Freeway at the intersection of Wenkes Road just before 10.30am, Thursday, August 31.
Victoria Police confirmed all four occupants of the sedan died at the scene.
"The driver of the truck, a 30-year-old man, sustained non life-threatening injuries and was conveyed to hospital," Leading Senior Constable Kendra Jackson said.
"Major Collision Investigation Unit detectives are en route to the scene.
"The exact circumstances surrounding the collision are yet to be determined and investigations remain ongoing."
Northbound traffic on the Hume Highway is closed at the Rutherglen-Springhurst Road.
Anyone who witnessed the incident, has footage or information is urged to contact Crime Stoppers on 1800 333 000 or make a report online at www.crimestoppersvic.com.au.
Emergency services are responding to a serious crash on the Hume Freeway at Chiltern, with drivers urged to avoid the area.
Crews were called to a crash, believed to involve a truck and at least one other vehicle, about 10.30am on Thursday, August 31.
Northbound traffic is being diverted at Springhurst.
Southbound traffic is not impacted.
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A Department of Transport spokesman said it's unclear how long the road would be closed for.
"Drivers heading towards Wodonga will be diverted onto Rutherglen-Springhurst Road then the Murray Valley Highway, to return to the freeway at Barnawartha North," the spokesman said. "Melbourne-bound lanes remain open.
"This incident and road closure is under the control of emergency services.
"Lanes will only re-open when it's safe to do so.
"VicRoads staff will provide assistance in setting up traffic management on detour routes."
More to come.
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