Slovakia ended Scotland's nine-match unbeaten 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.
Gregus scored his fourth goal for his country with a deflected shot in the 32nd minute to deny Scotland a chance to win League B Group 2, and promotion to the top league, with one match to spare.
Scotland, who won a penalty shootout against Serbia to qualify for Euro 2020, nearly equalised with the last kick of the game when Leigh Griffiths got in behind the Slovakia defence but goalkeeper Marek Rodak got his fingertips to the ball.
Scotland have 10 points from five games, four ahead of Czech Republic who host Israel later on Sunday. Slovakia, who beat Northern Ireland in extra-time on Thursday, are bottom with four points.