Maverick Vinales claimed his first pole since the opening round in Qatar, producing a superb late lap to deny Pol Espargaro in San Marino as Marc Marquez and Valentino Rossi were involved an incident that saw both men summoned to the stewards.
Espargaro had looked set to claim his maiden MotoGP pole and the first for KTM as he moved to the top of the timesheets with a lap of 1:32.560.
However, KTM saw a piece of history snatched away from them at Misano, where Vinales went 0.295 seconds quicker to take the top slot on the grid.
The Monster Energy Yamaha rider will now hope for a better result than he enjoyed in the season opener in Qatar. Vinales was left to settle for a seventh-place finish on that occasion despite starting at the front.
Qualifying specialist Fabio Quartararo completed the front row of the grid for Petronas Yamaha, with runaway championship leader Marc Marquez only able to claim fifth behind Franco Morbidelli after a tempestuous duel with Rossi.
Marquez was involved in a crash in the fourth practice session but appeared well positioned to challenge for the top three on his final flying lap.
However, he and Rossi traded overtaking moves before the Italian pushed the Spaniard wide at turn 14, the two riders coming close to making contact.
The near-collision hindered each rider's lap, with Marquez gesturing angrily at the seven-time premier class champion after being condemned to the second row.
It was later confirmed the pair would be required to attend a hearing with the stewards panel regarding the incident.
Rossi qualified seventh behind Andrea Dovizioso, who is Marquez's closest challenger in the championship, the Ducati rider trailing the Repsol Honda star by 78 points with a maximum of 175 points still to be claimed.
LCR Honda's Cal Crutchlow failed to make it out of Q1 and will start from 14th, with Aleix Espargaro just behind him for Aprilia. Espargaro's team-mate Andrea Iannone was unable to compete in qualifying after an FP4 crash left him nursing a shoulder issue that will keep him out of the race.
Jorge Lorenzo, himself still recovering from injury, could only qualify 18th.
1. Maverick Vinales (Monster Energy Yamaha): 1:32.265
2. Pol Espargaro (KTM): +0.295s
3. Fabio Quartararo (Petronas Yamaha): +0.306s
4. Franco Morbidelli (Petronas Yamaha): +0.445s
5. Marc Marquez (Repsol Honda): +0.477s
6. Andrea Dovizioso (Ducati): +0.773s
7. Valentino Rossi (Monster Energy Yamaha): +0.814s
8. Johann Zarco (KTM): +0.858s
9. Alex Rins (Suzuki Ecstar): +1.000s
10. Joan Mir (Suzuki Ecstar): +1.166s
11. Takaaki Nakagami (LCR Honda): +1.184s
12. Michele Pirro (Ducati): +1.196s