Slovakia ended Scotland's nine-match unbeaten run when a first-half goal from Jan Gregus gave them a 1-0 win in their Nations League match on Sunday between teams who both qualified for Euro 2020 amid drama last week.
In other League B matches, Wales kept their seventh successive clean sheet in competitive games as they beat Ireland 1-0 and Turkey hit back to beat 10-man Russia in a match which hinged on an early red card for Andrey Semenov.
Gregus scored his fourth goal for his country with a deflected shot in the 32nd minute to deny Scotland a chance to win Group B2, and promotion to the top league, with one match to spare.
Scotland have 10 points from five games, one ahead of Czech Republic who beat Israel 1-0 with a seventh minute goal by captain Vladimir Darida. Slovakia are bottom with four points.
David Brooks headed the winner for Wales who stayed top of Group B4 with 13 points from five games and need a draw at home to Finland, who are one point behind after winning 2-1 in Bulgaria, on Wednesday to win promotion to League A.
Teemu Pukki and Robin Lod gave Finland a 2-0 halftime lead before Bulgaria's Galin Ivanov had a penalty saved by Lukas Hradecky in the 61st minute.
Bulgaria won another penalty seven minutes later which substitute Dimitar Iliev converted and were unlucky not to get a draw.
Denis Cheryshev gave Group B3 leaders Russia an early lead but the visitors were left a man short in the 24th minute after a poor back pass by Magomed Ozdoev left Cenk Tosun with a clear run on goal.
Semenov unceremoniously brought him down and was sent off.
Kenan Karaman headed Turkey level almost immediately and Cengiz Under put them in front with a superb finish six minutes later. Tosun added a third with a penalty early in the second half before Daler Kuzyaev pulled one back.
Austrian substitutes Louis Schaub and Adrian Grbic scored in the last 10 minutes as they hit back to beat Northern Ireland 2-1 in Group B1 after Josh Magennis gave the visitors the lead in the 75th minute.
Schaub and Grbic came on three minutes later and combined for Schaub to equalise in the 81st minute before Grbic scored the winner from Marko Arnautovic's pass.
Austria lead with 12 points from five games, three ahead of Norway whose match away to Romania was cancelled after the team was quarantined and barred from travelling by Norwegian health authorities due to a positive COVID-19 test.
Norway's fate is in the hands of UEFA who could hand them a 3-0 defeat for a no-show.
(Reporting by Brian Homewood, editing by Ed Osmond, Toby Davis and Pritha Sarkar)
