Bezzecchi became the first rider to break the one minute and 30 seconds barrier, clocking 1:29.982 to give Aprilia pole on the team's home turf.
"It was a good lap; I'm happy because the qualifying wasn't easy. I met a lot of yellow flags , and it was difficult to find the concentration," Bezzecchi said.
"In the first stint I stayed on the track one lap more just to have a lap time , and fortunately it was my best one because in the second stint I couldn't have a clean lap."
The Italian complained by raising his hand when LCR Honda's Johann Zarco impeded him, and the stewards said they were investigating the incident.
Bezzecchi will be joined by Marquez and Gresini Racing's Fermin Aldeguer on the front row.
Marquez attempted to better his time twice late in the session but pulled out of both flying laps as he tried to ride over the limits on the turns.
"Last year my pace was a bit better, but this year it looks like Marco is super fast, extremely fast in the fast corners," Marquez said.
"Let's see if we can fight, but the target was the front row. When I stopped in the box, I saw the 1:29 (from Bezzecchi). I said, 'OK, ciao pole position, I will not arrive there because I knew where was my limits.'
"I overrode; I tried to push to 100%. It was enough to achieve the target, but not enough to be in pole position."
VR46 Racing's Fabio Di Giannantonio went fourth fastest while championship leader Jorge Martin was only fifth fastest and has Honda's Luca Marini alongside him on the second row.
Martin leads the championship with 256 points while Marquez is second (237 points) and Bezzecchi third (232 points).
KTM's Pedro Acosta, VR46 Racing's Franco Morbidelli and Gresini Racing's Alex Marquez - who crashed twice in the qualifying session - will start on the third row.
Francesco Bagnaia, who held the previous lap record at Misano, continued to struggle aboard his Ducati - as he has all season - and will start 14th on the grid after failing to advance from Q1 to Q2.
