The reigning world MotoGP champion clocked 1min 54.981sec on his Honda to finish 0.92sec ahead of Italian veteran Rossi, of Yamaha, with Spaniard Dani Pedrosa at 0.138 for Honda.
The 24-year-old Marquez’s 41st pole position in MotoGP, and 69th across the three classes, gives him a fantastic chance of extending his championship advantage.
Considering his domination of the last four seasons that have delivered three top-class titles, Marquez does not have a very good record around the Brno circuit and is without a win since 2013.
But he was in fine fettle in qualifying, and his rivals will have to find some extra speed to beat him in Sunday’s race.
The five-time world champion’s compatriot Maverick Vinales struggled though, and qualified down in seventh place as he looks to cut the five-point gap to Marquez at the top of the riders’ standings.
In the tightest title battle since the points system was changed in 1993, the 38-year-old Rossi is still in the hunt for a tenth world title.
The legendary Italian has only won once this season, but has remained consistent and is only ten points adrift of Marquez in fourth.
He produced an impressive lap in qualifying to take second place, after going third-fastest in both the third and fourth practice sessions earlier in the day.
The other world title contender, Ducati’s Andrea Dovizioso, showed reasonable speed on Saturday and will start the race from fourth place.
Briton Cal Crutchlow and former world champion Jorge Lorenzo complete the second row.
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