Jorge Lorenzo clinched his first pole position since round four of the MotoGP Championship as he put in a blistering 1:54.989, a new lap record.
All eyes were on the Yamaha following his record-breaking pace in FP3 in which he set a 1:55.476 at the Brno circuit.
The Spaniard, second in the Championship standings, set the early pace with a 1:55.238 while Marc Marquez lined up in second place, 0.311s off the pace.
In contrast it was a disappointing start to the session for their team-mates. Valentino Rossi's opening gambit was only good enough for seventh while Dani Pedrosa, who was riding in "quite intense pain" having hurt his foot in a massive highside on Friday afternoon, slotted into eighth place.
With Lorenzo and Marquez both in the pits for fresh rubber, Rossi upped his place to slide into fourth.
Heading out for a second run Lorenzo laid down his marker as he crossed the line with a 1:54.989, which was half a second up on Cal Crutchlow's pole position record.
Marquez tried to challenge but fell short by 0.074s while a late effort from Rossi saw him snatch third from Andrea Iannone.
Bradley Smith and Andrea Dovizioso completed the second row of the grid ahead of Maverick Vinales, Pol Espargaro and Pedrosa.
Earlier Espargaro and Cal Crutchlow were the top riders who advanced through Q1. The duo finished the afternoon eighth and 10th.
Meanwhile Claudio Corti, in action for Forward Racing who were back in MotoGP after their earlier woes, qualified P24.
Karel Abraham, who missed the last four rounds with a foot injury, will start his home race from the very back of the grid. He was 3.691s off the pace in Q1.
1 Jorge LORENZO Movistar Yamaha MotoGP 1'54.989
2 Marc MARQUEZ Repsol Honda Team 1'55.063 +0.074
3 Valentino ROSSI Movistar Yamaha MotoGP 1'55.353 0.364
4 Andrea IANNONE Ducati Team 1'55.390 0.401
5 Bradley SMITH Monster Yamaha Tech 3 1'55.460 0.471
6 Andrea DOVIZIOSO Ducati Team 1'55.935 0.946
7 Maverick VIÑALES Team SUZUKI ECSTAR 1'55.954 0.965
8 Pol ESPARGARO Monster Yamaha Tech 3 1'55.955 0.966
9 Dani PEDROSA Repsol Honda Team 1'55.969 0.980
10 Cal CRUTCHLOW CWM LCR Honda 1'56.192 1.203
11 Hector BARBERA Avintia Racing 1'56.399 1.410
12 Yonny HERNANDEZ Octo Pramac Racing 1'56.739 1.750
13 Danilo PETRUCCI Octo Pramac Racing 1'56.713
14 Scott REDDING EG 0,0 Marc VDS 1'56.765
15 Aleix ESPARGARO Team SUZUKI ECSTAR 1'56.806
16 Stefan BRADL Aprilia Racing Team Gresini 1'57.133
17 Mike DI MEGLIO Avintia Racing 1'57.215
18 Loris BAZ Forward Racing 1'57.540
19 Alvaro BAUTISTA Aprilia Racing Team Gresini 1'57.552
20 Eugene LAVERTY Aspar MotoGP Team 1'57.634
21 Nicky HAYDEN Aspar MotoGP Team 1'57.645
22 Jack MILLER CWM LCR Honda 1'57.855
23 Alex DE ANGELIS E-Motion IodaRacing Team 1'58.599
24 Claudio CORTI Forward Racing 1'59.145
25 Karel ABRAHAM AB Motoracing 1'59.903