Lorenzo went straight to the top of the timesheets on his first run, beating the lap record he had set during Saturday’s third practice session in the process.
The Yamaha rider then lowered the record again with a time of 1m54.989s, nearly half a second faster than anybody else had gone during the session.
Honda’s Marquez improved significantly on his second attempt, but ultimately fell just 0.074 seconds shy.
“Before qualifying, I thought a laptime similar to the time this morning would be good enough for pole position but I was wrong,” Lorenzo, the last non-Honda rider to win at Brno, said.
— Jorge Lorenzo (@lorenzo99) August 14, 2015
“Really I pushed more than the first time out because I had a very good laptime and I thought I would risk a little bit more – if I crash from the front it’s OK.
“I didn’t crash, I improved a little bit on braking and a little bit on throttle.
“Maybe the laptime was not perfect. In the last corner I opened maybe too much throttle, too much [wheel] spinning and lost some drive, maybe losing one tenth.
“But the rest of the lap I’m very proud of, I really made one of my best laps ever.”
Championship leader Valentino Rossi completed the front row on the second Yamaha as he slotted into third place with his final run of the session.
Rossi’s effort shuffled Ducati’s Andrea Iannone down to fourth place, just ahead of Tech 3 rider Bradley Smith.
Andrea Dovizioso, on the second Ducati, will line up sixth on the grid for Sunday’s 22-lap race ahead of Maverick Vinales and Pol Espargaro, who had earlier made it through as the fastest rider from Q1.
Dani Pedrosa will start ninth but the defending Brno winner was almost a full second slower than Lorenzo’s best time as he continues to suffer from an ankle injury sustained on Friday.
Cal Crutchlow completed the top 10 on his LCR Honda, but the British rider had earlier been forced to come through Q1 along with Pol Espargaro.
Hector Barbera and Yonny Hernandez were the slowest riders in Q2 and will start the race from 11th and 12th.