Jan Vertonghen will play no further part in Euro 2016 after tearing his ankle ligaments in training.
Belgium coach Marc Wilmots revealed in a press conference on Thursday evening that Vertonghen tore multiple ligaments in his ankle just before he blew the whistle to end the training session.
Wilmots confirmed Vertonghen's injury will rule him out of the rest of Euro 2016 and expects the 29-year-old to be sidelined for a period of six to eight weeks, meaning he could miss the start of the Premier League season with Tottenham.
Wilmots said: "He turned his ankle. He had a scan here at Lille and he has torn two of his three external ligaments in his ankle which means his European Championships is over.
"I'm not going to talk about a tragedy because there are far more tragic things in life but it is a sporting tragedy for him. He was a very important player for us.
"Mentally and physically he was completely fit and he had a a lot of desire but unfortunately on a specific movement he has done 10,000 times, he turned his ankle."
Vertonghen tweeted: "Gutted to say that my Euros is over. Good luck to my team-mates for the rest of the competition."
The loss of Vertonghen, who has been playing at left-back during the tournament, is a further blow for Wilmots, who will also be without suspended Barcelona centre-half Thomas Vermaelen for the game against Wales.
Vertonghen had been an option to slide into centre-back to resume his hugely successful club partnership alongside Totttenham team-mate Toby Alderweireld but that role is now likely to be given to 21-year-old Jason Denayer.
The inexperienced Jordan Lukaku, 21, is the likely candidate to deputise at left-back. He currently plays for KV Oostende in the Belgian Pro League.
Belgium are already without captain Vincent Kompany, who missed the entire tournament with a thigh injury, as did fellow defenders Nicolas Lombaerts and Dedryck Boyata.
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Wales manager Chris Coleman was asked how much Vertonghen's injury might hurt Belgium in his Thursday press conference but he insisted his team must focus on themselves.
"He's a good player, an outstanding performer, if he misses the game it's a big loss, but they have so much talent, but we can't look past ourselves," Coleman told reporters on Thursday afternoon.
"We only concentrate on ourselves. And we need to do that. We need to ensure there's a high level or urgency about us. Once we get that right we know we can be in the game. I don't know who is available, I just know about us."
There was better news elsewhere on the injury front for Belgium as captain Eden Hazard returned to training after missing practice sessions on Tuesday and Wednesday because of a thigh problem.
The Chelsea star trained early Thursday at the team's base in Bordeaux, before the Belgians' scheduled departure to the northern city of Lille where Friday's match against Wales is staged.