Gareth Bale has been ruled out of Wales' crucial World Cup qualifying double-header next month with what boss Rob Page describes as a "significant hamstring tear".
The 32-year-old has not featured for club or country since sustaining the injury during a training session with Real Madrid three weeks ago.
Madrid boss Carlo Ancelotti recently said he is unsure how long Bale will be out but there was hope he would be back in time for Wales' trips to the Czech Republic and Estonia.
However, Bale – who is one cap short of a milestone 100 for Wales – was left out of Page's squad on Tuesday and is also a doubt for his side's final three qualifiers in November.
"Hamstring injuries are graded from one to four and this is a four. It's a significant hamstring tear," Page said at a news conference.
"This camp has come too soon for him. He's not going to be involved, and that may also be the case progressing to November's camp."
Bale started Madrid's opening three games of the season and featured in all three of Wales' matches last month, scoring a hat-trick in the 3-2 win away at Belarus.
That ended a 17-game drought without a goal for the Dragons – his second-longest such run – but he could not find a way through in the goalless draw with Estonia last time out.
Wales are third in Group E after four matches, one point behind next opponents the Czech Republic with a game in hand as the pair fight for second place behind runaway leaders Belgium.
Page's side will head into that clash in Prague without undisputed star player Bale, who has been involved in all five of his side's goals this campaign (three goals and two assists).
"He's been first class for Wales over the years, so of course it's a blow for myself and the supporters," Page said. "But it does give others a chance.
"We can't cry about it. Gareth showed his worth in the last camp and he was at another level.
"We've got new players coming through, and an abundance of players who can fill the position he plays. There aren't many Gareth Bales in the world but others will have a chance."
Juventus midfielder Aaron Ramsey is back involved for Wales after withdrawing from the last squad through injury.
Ramsey has featured four times in Serie A this term and leads the way for passes per 90 minutes (67.71) and successful passes (61.71) among Juventus midfielders.
He has missed several key fixtures for Wales this campaign, but Page insists the former Arsenal man's commitment cannot be questioned.
"We're confident he'll be with us," Page said.
"I understand the fans' frustration because they want the best players turning up. He can put a shift in and did a full pre-season with Juventus.
"Players like him are prone to picking up injuries. He's managing his body better now and I think we're seeing the best of it.
"He's become more robust and shown over the summer he's more capable of playing games. His training will be adapted but we expect him to play both games."