Liberal MPs elected Mr Pesutto to replace Matthew Guy in a secret ballot during a party room meeting on Thursday morning, beating out Berwick MP Brad Battin.
David Southwick has been re-elected deputy leader, shadow health spokeswoman Georgie Crozier will be upper house leader and transport infrastructure spokesman Matt Bach her deputy.
Mr Pesutto narrowly reclaimed Hawthorn after he lost the inner east seat the 2018 state election.
He faces a steep challenge after the party secured just 29 per cent of the primary vote at the November poll, its worst result in decades.
Before the vote, Mr Pesutto said the party would rally behind the new leader regardless of the result.
"We're going to work hard in a unified way," he said.
"We're going to make sure that the Victorian people see an opposition that holds the government to account and that is ready to govern."
It is Mr Battin's second failed tilt at the Liberal leadership after challenging Michael O'Brien in March 2021.
Richard Riordon and Ryan Smith threw their support behind him after withdrawing from the leadership race.
Mr Guy has not revealed if he intends to leave parliament after suffering his second consecutive loss to Premier Daniel Andrews, and declined to speak to media when arriving at parliament.
Mr Guy was defeated in the 2018 "Danslide" and resigned his position to serve as a backbencher. He then reclaimed the leadership from Malvern MP Michael O'Brien in 2021.
Labor has won at least 54 seats, the coalition 27 and the Greens four in the lower house
Late on Wednesday, the Victorian Electoral Commission declared Labor the winner of Preston in Melbourne's north by more than 1600 votes after preferences over the Greens.
The results for Bass and Pakenham are still unknown but are expected to be finalised on Thursday.
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