England has lost a qualifying game for the World Cup or European Championship for the first time in 10 years.
Substitute Zdenek Ondrasek scored his first international goal to rally the Czech Republic to a 2-1 win over England in a Euro 2020 qualifier on Friday.
Captain Harry Kane put England ahead in the fifth minute from the penalty spot.
A victory would have secured a spot for England at the finals with three games left.
Jakub Brabec equalized for the Czechs four minutes after Kane’s goal and Ondrasek scored the winner five minutes from time with a low right-footed shot from inside the area.
England and the Czech Republic top Group A with 12 points but England has one game in hand.