Newcastle United winger Ryan Fraser is set to miss out for Scotland against Serbia on Thursday.
The Scots are involved in a Euro 2020 playoff, which they must win to progress to the tournament in the summer.
Now it is revealed that both Fraser and Grant Hanley will be out of the game.
Fraser has a hamstring issue, which he picked up before his team's 2-0 loss to Southampton on Friday.
"It's disappointing," said Scotland assistant manager Steven Reid to reporters.
"Grant's missed a little bit of football and unfortunately won't be with us, and Ryan, who played really well in the last camp.
"That is a blow, but we've got strength in depth. We've obviously got bigger numbers than usual in the squad [due to the Covid-19 situation] and we feel we've got a squad capable of competing at our strongest in the Serbia game and then in the two Nations League games after [against Slovakia and Israel].
"What's been pleasing in the last couple of camps is that despite the issues and the injuries and the Covid situation, the lads who stepped up were first class."