Czech Republic stunned England with a late Zdenek Ondrasek strike to earn a 2-1 win in Euro 2020 qualification on Friday (Saturday AEDT) to pull level with their opponents on 12 points at the top of Group A.
Turkey snatched a late 1-0 win over Albania to maintain their lead at the top of Group H, ahead of France on head-to-head ranking, after Les Bleus beat Iceland 1-0.
Ukraine moved to the verge of qualification for the tournament with a 2-0 win over Lithuania to maintain their five-point lead at the top of Group B.
Portugal are in second place in the group after they beat Luxembourg 3-0 at home.
In Prague, Harry Kane sent England ahead from the spot after Raheem Sterling was brought down by Lukas Masopust in the area.
The Tottenham forward sent Sevilla goalkeeper Tomas Vaclik the wrong way to break the deadlock after five minutes.
However Czech Republic equalised four minutes later when Jakub Brabec stole in at the back post to score after a corner was flicked on.
The Czechs had the better of the encounter and deserved to end England's 42-game run without defeat in qualifying matches.
Ondrasek, on for his debut at the age of 30, tucked home Lukas Masopust's cross to earn an impressive victory for the hosts.
"Tonight we didn't do enough to win the game," England manager Gareth Southgate told ITV.
"There are things we can look back on, but collectively we have to accept that there weren't enough good performances."
Montenegro drew 0-0 with Bulgaria in the group's other match.
France were set to move top of Group H after Olivier Giroud's penalty scraped victory over Iceland, but Cenk Tosun's 90th minute strike helped Turkey edge Albania in a cagey clash.
Both Turkey and France sit on 18 points from seven matches, while Iceland have 12 leaving qualification unlikely for the Euro 2016 surprise package.
In the other match in the group, Andorra beat Moldova 1-0, leaving them both on three points.
Ukraine made light work of Lithuania to virtually assure their progress to next northern summer's tournament, dominating the clash in Kharkiv.
Ruslan Malinovskyi struck once in each half, with Taras Stepanenko and Andriy Yarmolenko hitting the woodwork.
Atalanta playmaker Malinovskyi's brilliant dribble and one-two saw him break into the area and he finished with aplomb in the 29th minute.
His second was even more spectacular, a free-kick which left Lithuania goalkeeper Vytautas Cerniauskas rooted to the spot.
In the other Group B clash, Portugal took control of the race for second with a comfortable win over Luxembourg which saw them rise to 11 points, four clear of third place Serbia.
Manchester City winger Bernardo Silva broke the deadlock in the 16th minute and Cristiano Ronaldo netted the second after the break with a perfect lob after stealing the ball back high up the pitch.
Goncalo Guedes notched the third in Portugal's impressive triumph in Lisbon, which leaves their opponents out of contention for qualification.
Australian Associated Press