Superintendent John Fitzpatrick said the crash, which occurred at Brimin just before 10pm on Friday, was devastating.

"The gravity of that scene. To see a family in that position. It was very difficult for the emergency service workers," he said.

He said the driver of the truck, who stayed at the scene following the crash, was yet to be interviewed, but police believe he may have veered onto the wrong side of the road.

"Part of the investigation will investigate whether distraction, fatigue, speed, or drugs, are a part of this. With the passage of time, those sorts of things will be revealed," Superintendent Fitzpatrick said.

The scene of the collision, about 500 metres west of Brimin Road, was closed to traffic as detectives from the major collision investigation unit investigate the cause of the crash.

It was the second fatal crash on Victoria's roads in the lead-up to the Melbourne Cup long weekend.

A motorcyclist died after colliding with a Mitsubishi Outlander on the Princes Highway in Pakenham in Melbourne's south-east about 6.50am on Friday.

The dark grey Mitsubishi made a U-turn before colliding with the motorcycle, police said. The 28-year-old male motorcyclist was flown to hospital, but later died.

On Saturday morning, another motorcyclist was killed in Yarrambat, north-east of Melbourne.

The rider, believed to be a man in his 30s, lost control of the bike and crashed on Ironbark Road about 9.30am, police said.

Superintendent Fitzpatrick urged drivers to be cautious on the roads.

"To have lost four people means the message is not getting through."

Victoria's country roads, which claimed 151 lives last year, featured on the state election campaign trail earlier this week.

Opposition Leader Matthew Guy said he would fix country roads "once and for all", with $1 billion for upgrades over 10 years, if the Coalition is elected on November 24.

However, Premier Daniel Andrews dismissed the Coalition's promise, saying his government had set aside $941 million for country roads in this year’s budget.

South-West Coast MP Roma Britnell said the state of country roads was the most pressing issue people raised with her.

"Our roads are falling apart. When they do fix them they fix them poorly," the Liberal MP said.

Last year, eight people died on Victoria’s roads over the Melbourne Cup long weekend.

Superintendent Fitzpatrick urged anyone driving long distances to slow down, turn off their phone and take regular breaks.

"We don’t want to see anyone gambling with their life this long weekend."

Anyone who witnessed any of the collisions should contact Crime Stoppers on 1800 333 000.