Virginia Raggi's triumph in Rome made her the first female leader of the capital in its more than two and a half millennia of existence.
And it was a win based on exactly the kind of pro-change platform and mood that had, until now, underpinned Prime Minister Matteo Renzi's popularity with an electorate that has grown weary of its political class after a decade and a half of economic stagnation.
Worryingly for the premier's Democratic Party (PD), the landmark victories were achieved by M5S securing the backing or the abstention of anti-Renzi forces encompassing the far-left, the far-right and the mainstream centre-right.
"Losing Rome and Turin is a blow, it's painful," acknowledged PD president Matteo Orfini.
Analysts concurred that Italy's political landscape had been redrawn with potentially major repercussions for an October referendum on constitutional reform and a general election due by 2018.
Renzi has vowed to quit if he loses the October vote on streamling Italy's parliamentary and electoral systems.
"The bottom line is that the PD is losing ground in favour of anti-establishment parties," said economist Lorenzo Codogno.
He said the vote also marked the emergence of a "more hands-on and pragmatic" breed of M5S politician which could make the party more electable in 2018.
"The Italian political scene has become a tri-party system in which Five Star is taking the lead as the major opposition party."
'New era opens'
Both the new M5S female mayors are successful career women from well-heeled backgrounds.
They ran campaigns which largely avoided national issues such as the party's support for a referendum on leaving the eurozone.
And their sober style contrasts markedly with the shrill tone of M5S's acerbic figurehead and founder, comedian Beppe Grillo.
The victory in Rome surpassed all expectations with 37-year-old lawyer Virginia Raggi taking 67 percent of the votes cast in a run-off against the PD's Roberto Giachetti.
In Turin, where the anti-immigrant, anti-EU Northern League is an influential player in local politics, Chiara Appendino, 31, claimed just over 54 percent to oust long-serving PD heavyweight Piero Fassino.
The only consolation for Renzi was that the centre-left held on to Milan, where the former World Expo director Giuseppe Sala squeezed home in a more traditional fight with a centre-right rival. PD-allied candidates also claimed anticipated wins in Bologna and Naples.
"For the first time Rome has a female mayor in an age where equality of opportunity remains a mirage," Raggi said in her victory speech.
"I will be a mayor for all Romans. I will restore legality and transparency to the city's institutions after 20 years of poor governance. With us a new era is opening."
Appendino, a multilingual businesswoman who helps run her family's laser equipment company, struck a similar note.
"We have made history," she said. "This was not a protest vote, it was about pride and change."
Raggi was virtually unknown a few months ago and has no experience of running anything.
But she ran a shrewd campaign that tapped into deep reserves of anger among voters over the dilapidated state of the capital's streets, public transport and garbage management.
Her cause was helped by the ousting of her predecessor, the PD's Ignazio Marino, over an expenses issue and a much bigger scandal over organized crime's infiltration of the city administration.
Raggi has vowed to make such abuses impossible and get public services back on track.
But many analysts are sceptical about her ability to make a difference given the city's huge structural problems, which include debts of over €13 billion ($14.7 billion).
The new mayor was a late entrant to politics, telling AFP recently that she had been inspired by the birth of her son Matteo to try and make her home city a better place for him to grow up in.
One area in which she is likely to make an immediate impact is on the credibility of Rome's bid to host the 2024 Olympics - one of Renzi's pet projects.
She said during the campaign that the city should address its problems first, then think about an Olympic bid.