Test rider Stefan Bradl will replace the Spaniard for the race.
Marquez had fresh surgery on Monday to replace a damaged titanium plate inserted after he broke his right arm in the 19 July season-opening race in Spain.
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His team had said that the 27-year-old, who tried to return for the second race in Jerez but withdrew without qualifying, would stay in the hospital in Barcelona for 48 hours before being discharged.
“After a heroic return just four days after surgery in Jerez, Marc Marquez will miss the Brno race, with Stefan Bradl joining the Repsol Honda Team alongside Alex Marquez,” Repsol Honda said in a statement.
“After undergoing a second operation… Marquez and HRC have decided the world champion will not ride in the Czech Republic in order for him to recover more.
“In his place, HRC test rider Stefan Bradl will mount the Honda RC213V.”
Bradl has claimed three top-10 finishes at Brno from his seven starts in the MotoGP class and only once missed out on points.