Marquez beat Trackhouse rider Ai Ogura while Francesco Bagnaia was third on the other factory Ducati.
Ogura led the first lap from pole position and was pressured early by VR46 rider Fabio Di Giannantonio from second place.
However, the satellite Ducati rider faded in the opening laps before recovering to finish fourth.
From third on the grid, Bagnaia dispatched Ogura on Lap 2 and fourth place starter Marquez followed suit, having inherited a place at the start with Bezzecchi absent.
Bagnaia controlled the opening stanza until Lap 15 when Marquez made his move for the lead on the Italian.
Bagnaia was left to battle over second and ultimately lost out to Ogura, who threatened Marquez’s lead in the closing laps.
Secure your spot today.
Ultimately, the Spaniard clinched victory by a slender 0.421s while Bagnaia narrowly held onto third from Di Giannantonio.
“I’m extremely happy with this win,” said Marquez.
“It was somehow an unexpected one, as I was sure that I would struggle towards the end.
“It did happen, actually, but the others were not quicker than me.
“I believe that the bike had something more, but I wasn’t able to make the most of it, especially in the change of direction between Turns 6 and 7, which is normally one of my strong points, but where I was instead losing time.
“I’m taking each Grand Prix as it comes and right now I’m enjoying the moment. We’ll have a test tomorrow and then head straight to Assen.”
Bezzecchi was absent from the Grand Prix of Czechia after he was given a one-race ban for abusing a marshal.
With no points in either Saturday’s Sprint or Sunday’s Grand Prix, Bezzecchi’s lead has been whittled down to just eight points over his Aprilia teammate Jorge Martin.
Martin finished the feature race in ninth after serving a double-long-lap penalty for the pile-up he caused in the preceding Grand Prix of Hungary at Balaton Park.
“It was a difficult weekend – with Jorge’s double long-lap penalty in the main race and Marco’s disastrous Saturday, which even deprived us of the chance to see him race,” said Aprilia boss Massimo Rivola.
“The standings were not our focus before, and they are not our focus now. We must react as a united team and as the strong company that we are, and keep working to improve the bike.
“There’s still a long way to go in the championship, and our rivals have clearly taken a step forward – we must do the same.
“Congratulations to Ai Ogura, who had an extraordinary weekend with a pole position, a new lap record and two podium finishes. This result demonstrates the competitiveness of the RS-GP26.”
There’s no rest for MotoGP with the world championship resuming at Assen for the Grand Prix of the Netherlands on June 26-28.
Results: MotoGP Grand Prix of Czechia, Brno Circuit
| Pos | Num | Rider | Team | Bike | Laps/Diff |
| 1 | 93 | Marc Marquez | Ducati Lenovo Team | Ducati | 21 laps |
| 2 | 79 | Ai Ogura | SuperFile Trackhouse MotoGP Team | Aprilia | 0.421 |
| 3 | 63 | Francesco Bagnaia | Ducati Lenovo Team | Ducati | 2.255 |
| 4 | 49 | Fabio Di Giannantonio | Pertamina Enduro VR46 Racing Team | Ducati | 2.424 |
| 5 | 36 | Joan Mir | Honda HRC Castrol | Honda | 12.810 |
| 6 | 54 | Fermin Aldeguer | BK8 Gresini Racing MotoGP | Ducati | 14.874 |
| 7 | 25 | Raul Fernandez | SuperFile Trackhouse MotoGP Team | Aprilia | 18.657 |
| 8 | 10 | Luca Marini | Honda HRC Castrol | Honda | 21.265 |
| 9 | 89 | Jorge Martin | Aprilia Racing | Aprilia | 21.401 |
| 10 | 23 | Enea Bastianini | Red Bull KTM Tech3 | KTM | 22.273 |
| 11 | 11 | Diogo Moreira | Pro Honda LCR | Honda | 22.881 |
| 12 | 33 | Brad Binder | Red Bull KTM Factory Racing | KTM | 22.942 |
| 13 | 21 | Franco Morbidelli | Pertamina Enduro VR46 Racing Team | Ducati | 25.003 |
| 14 | 7 | Toprak Razgatlioglu | Prima Pramac Yamaha MotoGP | Yamaha | 25.806 |
| 15 | 12 | Maverick Viñales | Red Bull KTM Tech3 | KTM | 26.36 |
| 16 | 43 | Jack Miller | Prima Pramac Yamaha MotoGP | Yamaha | 33.121 |
| 17 | 35 | Cal Crutchlow | Castrol Honda LCR | Honda | 44.784 |
| DNF | 37 | Pedro Acosta | Red Bull KTM Factory Racing | KTM | Lap 20 |
| DNF | 42 | Alex Rins | Monster Energy Yamaha MotoGP Team | Yamaha | Lap 17 |
| DNF | 20 | Fabio Quartararo | Monster Energy Yamaha MotoGP Team | Yamaha | Lap 1 |

























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