Marquez resisted a strong challenge from the Ducatis of Andrea Dovizioso and Andrea Iannone in Brno MotoGP qualifying, as Yamaha had a disastrous session.
At a track that had been tipped as one of its best opportunities to unseat Marquez and Honda, Yamaha's first blow came when Valentino Rossi had a violent crash in practice four, falling moments before Alvaro Bautista did likewise at the same spot.
Rossi, who still ended the session fastest, required a precautionary x-ray on his left hand in the medical centre before being cleared to ride again. He did not suffer any broken bones, but did sustain a nasty cut to his little finger.
While Rossi was in pain, his team-mate Jorge Lorenzo seemed unable to get the Yamaha handling to his liking. Only a last-gasp lap got him up from 11th to sixth, one place ahead of Rossi.
With Yamaha struggling, Ducati emerged as Marquez's main threat.
Marquez had taken provisional pole early in Q2, but was then unseated by Pramac rider Iannone in the closing minutes.
The world champion's first reply could only get him to within 0.003 seconds of the satellite Ducati man, although Marquez then claimed pole back with a 1m55.585s.
Iannone could not respond, and was edged out of second by factory Ducati counterpart and 2015 team-mate Dovizioso, who slipped ahead by 0.012s. That still gave Ducati two bikes on the front row.
As had been the case in practice, Tech 3's Bradley Smith was the top Yamaha, thrusting himself ahead of Dani Pedrosa's Honda for fourth with his last lap.
Stefan Bradl and Yonny Hernandez made it through from Q1 to take eighth and 12th on the grid, sandwiching the Espargaro brothers and Cal Crutchlow.
Scott Redding couldn't repeat his Indianapolis Q2 appearance, due in part to a fall at the end of the practice session. He was fifth in Q1, just behind Gresini team-mate Alvaro Bautista's Factory-spec bike.
Mike di Meglio failed to set a qualifying time at all after crashing both his bikes - one in practice four and one at the start of Q1.
That wasn't what the Avintia crew needed after two accidents from di Meglio's team-mate Hector Barbera earlier in the weekend.
Britain's Leon Camier will start 18th for his second appearance standing in for Nicky Hayden at Aspar, while Colin Edwards' Forward replacement Alex de Angelis will begin his MotoGP return from 20th on the grid.
1. Marc Marquez (Spain) Honda 1:55.585
2. Andrea Dovizioso (Italy) Ducati 1:55.714
3. Andrea Iannone (Italy) Ducati 1:55.726
4. Bradley Smith (Britain) Yamaha 1:55.730
5. Dani Pedrosa (Spain) Honda 1:55.812
6. Jorge Lorenzo (Spain) Yamaha 1:55.815
7. Valentino Rossi (Italy) Yamaha 1:55.821
8. Stefan Bradl (Germany) Honda 1:55.871
9. Pol Espargaro (Spain) Yamaha 1:55.899
10. Aleix Espargaro (Spain) Yamaha 1:56.090
- Sports & Recreation
- Andrea Iannone
- Andrea Dovizioso
- Valentino Rossi
- Alvaro Bautista