Gareth Bale will miss Wales' final two World Cup qualifiers against Georgia and the Republic of Ireland.
Bale was ruled out following talks between the Football Association of Wales and his club Real Madrid and the result of a scan on his injured calf.
Barnsley striker Tom Bradshaw has been called up to replace Bale.
"The Football Association of Wales can confirm that Gareth Bale will miss the upcoming World Cup qualifiers against Georgia and Republic of Ireland," said an FAW statement
"Bale - who missed Real Madrid's game against Espanyol on Sunday - arrived with the rest of the Wales squad on Sunday and attended Monday's annual FAW Awards dinner at the team headquarters in Cardiff.
"But following dialogue with Real Madrid, Bale was sent for a scan and results confirmed that he wouldn't be fit to feature."
Bale's fitness has been a source of concern for Wales since he suffered a calf muscle strain against Borussia Dortmund in the Champions League last week.
Real boss Zinedine Zidane reported last weekend that Bale was suffering from "muscle fatigue", but added it was "nothing important".
"He could have played with us (against Espanyol) but I preferred to leave him out as he had a small complaint," Zidane said on Sunday.
"He's fine. After so many games at the start of the season and four months of injury, he was suffering after the game in Dortmund."
But Wales' worst fears were confirmed when the scan ruled out Bale of the Georgia game in Tbilisi on Friday and the visit of the Republic to Cardiff three days later.
Wales are currently in second place in Group D, four points behind leaders Serbia and one point above the Republic.
Two wins are likely to guarantee them at least a place in next month's World Cup play-offs.
Bale's absence could open the door for Liverpool teenager Ben Woodburn to make his first Wales start.
The 17-year-old made a dramatic introduction last month when he scored the winner within five minutes of coming on as a second-half substitute against Austria.
Three days later Woodburn made another huge impact from the bench when he set up the first goal in Wales' 2-0 win in Moldova.