The official MotoGP website rightly described it as 'Misano Mayhem' as changeable conditions led to a number of pit stops and tire changes with two-time champion Marquez making the correct late call on his Repsol Honda.
A switch to slick tires on a drying track saw him top the podium ahead of Bradley Smith and Scott Redding, the first time a British pair had been on the podium at this level since 1979.
Rossi, cheered to the echo by the 92,000 fans packed into his home circuit, could only manage fifth place, but his nearest title challenger, Spain's Jorge Lorenzo, crashed out when in second place.
It left the legendary Italian, seeking his 10th world crown, with 247 points, 23 points clear of his Movistar Yamaha teammate Lorenzo, with Marquez tracking them on 184.
Lorenzo, looking to close the gap on Rossi, suffered a minor injury to his hand when going off on Turn 15 with seven laps to go.
Marquez was able to ride to the finish with a solid advantage, claiming his fourth win of 2015 and 23rd overall.
From the start, the battle had been between the top three in the championship, but Marquez explained how pitting that bit earlier than Rossi and Lorenzo worked to his advantage.
"I was close behind the Yamahas to try and see how the asphalt and tires were," he told the official MotoGP website.
"I saw Lorenzo and Rossi had a lot of degradation and then I saw my bike started to move and I was losing so much time so I thought 'OK, it's time to change'. "
Rossi, while disappointed not to be able to delivery victory before his adoring fans, could look on the bright side.
"The result is good for the championship. I wanted to try to do a better race to arrive on the podium but I wasn't able to."
With five rounds of the 2015 season remaining, he remains in pole position with the action resuming in a fortnight in Spain.