Gareth Southgate made three changes from the crazy 5-3 win over Kosovo last month as Mason Mount was handed his first start at the expense of Ross Barkley.
Kieran Trippier got the nod over Trent Alexander-Arnold and Danny Rose started ahead of Ben Chilwell at left-back.
The Three Lions, who had not lost in 43 qualification games since being defeated by Ukraine in a World Cup qualifier in 2009, made the ideal start as they took the lead in the fifth minute.
Raheem Sterling beat the Czechs offside trap and raced into the area where he was brought down by Lukas Masopust as he tried to check back inside. The referee pointed to the penalty spot and Harry Kane did the rest going straight down the middle.
The lead only lasted four minutes as Jordan Pickford produced a fine one-handed save to deny Ondrej Celustka and conceded a corner. England failed to deal with the subsequent set-piece which was flicked on by Celustka and Jakub Brabec diverted the ball into the bottom corner past Rose at the back post.
The goal signaled a good spell of pressure from the hosts as England were at sixes and sevens defensively and struggled to get out of their own half, while the displayed very little as an attacking force.
Patrik Schick guided a header wide at the back post, while Celustka sent a header straight at Pickford from 15 yards and Jan Boril had a shot on the run saved as the Czechs pressed for a second goal.
England produced their best move of the game on 58 minutes when Kane’s reverse pass played in Sterling, but keeper Tomas Vaclik managed to get his hand to the ball to prevent the Manchester City forward putting the Three Lions back in front.
Czech Republic responded quickly and Pickford had to be alert to tip a stinging 20-yard effort from Masopust over the crossbar.
Rose escaped a penalty appeal when Vladimir Darida went to ground in the England area on 74 minutes, while Sterling appeared to be obstructed in the opposite box moments later, but again the referee waved play on.
On 77 minutes Alex Kral exchanged passes with Darida and got a sight of goal down the right, but his effort from an angle was pushed behind by Pickford.
Substitute Ross Barkley produced a sublime pass to play in Kane down the right of the area on 81 minutes, but Vaclik raced off his line to block the shot.
The Czechs won it in the 85th minute when Masopust got free down the right and pulled the ball back to the unmarked Zdenek Ondrasek in the centre and he made no mistake with a low right-foot strike past Pickford on his international debut.
The Czechs move level on 12 points with England, who have a game in hand, while Kosovo sit third another four points adrift.