The collision involving two SUVs occurred about 7.20am on Sunday on Bolinda-Darraweit Road in the Macedon Ranges, northwest of Melbourne.
The triple fatality brought the number of lives lost on Victorian roads in 72 hours to six, prompting renewed warnings from authorities.
Three people travelling in one SUV died at the scene, while two others involved in the crash were taken to hospital, one of whom had life-threatening injuries.
A dead kangaroo was found near the scene, with police looking into whether one of the SUVs had veered to avoid it.
"It appears one of the vehicles has veered into incoming traffic ... and they've collided head-on, which has caused the catastrophic scene," Senior Sergeant Ben Morris told reporters.
"We're not 100 per cent sure ... we're still working through the scene to confirm the involvement of the animal."
A motorist died hours later after a head-on collision on the Western Highway in Buangor, about 181km west of Melbourne.
Police were called to the scene after a car reportedly veered onto the wrong side of the freeway and collided with another vehicle.
The driver of the first car died at the scene and has yet to be formally identified.
The second car's driver was taken to hospital with non-life-threatening injuries, while a female passenger was airlifted to hospital with serious injuries.
In a separate incident on Saturday night, a rider died and another is fighting for life after a crash involving two motorcycles and two vehicles on the Western Freeway at Grangefields, near Melbourne.
A 16-year-old boy died in the city's east on Friday after being hit by a car, while an e-bike rider was critically injured in Geelong.
Road Policing Command Superintendent Justin Goldsmith said most of the serious collisions at the weekend were on regional roads.
"Every life lost is one too many," he said.
"What we have seen this weekend has been devastating and there are many families whose lives have changed forever as a result of the trauma on our roads."
Ambulance Victoria CEO Jordan Emery described the fatalities and injuries as the most horrific of circumstances.
"As a paramedic, I realise that these events are part of our job, but the tragic loss of life through road trauma is especially tough," he said."If there is one message we can send, it's to please take care when on the road. The impact is felt well beyond those behind the wheel."
There have been 156 lives lost on Victorian roads this year, 15 more than the same time in 2024.
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