The Three Lions took the lead in the Group A tie through a Harry Kane penalty but the hosts overturned the score courtesy of goals from Jakub Brabec and substitute Zdenek Ondrasek.
It is the first time Gareth Southgate’s side have dropped points in the section and they are now level with the Czechs at the summit having played one game fewer.
Trent Alexander-Arnold and Joe Gomez were unused substitutes in Prague.
Meanwhile, Reds striker Rhian Brewster came on at half-time for England U21s as they drew 2-2 with Slovenia away from home in a friendly.