Joe Allen has been ruled out of Wales' opening World Cup game against the United States on Monday.
The Swansea midfielder has not played since 17 September because of a hamstring injury and has been battling to be fit for Wales’ first game at the World Cup for 64 years.
Wales manager Robert Page said: "If I’m honest he’s not going to make it.
"We’d have liked him to play in the first game. But we’re not going to push him and hope that he will make the second game (against Iran on Friday)."
James Maddison appears to be a major doubt for England's World Cup opener after missing training on the eve of the Iran clash.
Gareth Southgate’s side kick-off Group B at the Khalifa International Stadium on Monday.
Maddison was the only player absent from training at England’s Al Wakrah Sports Complex base on Sunday afternoon.
The 25-year-old missed the Three Lions’ previous two open training sessions on Thursday and Friday due to what the Football Association called load management.
Maddison went off in the first half of Leicester’s win against West Ham last weekend with a knee complaint but said scans on the issue were fine.
"It’s OK," Maddison said on Wednesday. "We had a scan the day before we met up.
"It was a positive scan. It wasn’t any major problems. I have to do a bit of work to get up to speed with the physios here but it should be no concern."
Asked if the issue will impact his tournament, Maddison said: "I hope not, no.
"We’ve got to tick a few boxes with the physio team, there might be a bit of separate work, extra work to make sure it’s right and not a problem because I want to be fully, 100% if called upon. I’m hopeful I will be fine."
England’s remaining 25 players trained, including Kyle Walker and Kalvin Phillips as they work their way back to full fitness.
Australia winger Martin Boyle was ruled out of the World Cup by a knee injury on Sunday, two days before the Socceroos open their campaign against world champions France.
The Scottish-born forward, who had been an injury doubt since the squad arrived in Qatar after suffering the injury playing for his club Hibernian, will be replaced by Marco Tilio.
"We all feel for Martin and it is a cruel blow for him on the eve of the World Cup," coach Graham Arnold said in a statement.
"He has been an integral part of our journey to get to Qatar and we thank him for everything he has given the Socceroos during this campaign.
"He will be back and we can't wait to see him in the green and gold again next year."
Tilio, a 21-year-old winger, is in Qatar having arrived on Tuesday as precautionary cover for Boyle.
"Marco has done everything that has been asked of him to be ready for this tournament," Arnold said.
"He was an important member of our Tokyo 2020 Olympic team ... and he is familiar with the national team set up and we are looking forward to what he can bring to our squad for the tournament."
Australia also play Denmark and Tunisia in Group D.
France coach Didier Deschamps has decided not to replace injured striker Karim Benzema in his squad, leaving the reigning champions with 25 players for their title defence.
Benzema trained with the squad for the first time since arriving in Qatar on Saturday but was forced to leave the session early with a thigh injury and scans ruled him out of the tournament.
Asked on Sunday by TeleFoot whether he would replace the Ballon d'Or winner in his squad, Deschamps said "No".
"This is a quality group," he added. "In everything they do, both on and off the pitch, they are united. I have confidence in them."
Benzema was the second French player to join the casualty list in less than a week after forward Christopher Nkunku was injured in a training session and replaced in the squad by Randal Kolo Muani.
Influential midfielders Paul Pogba and N'Golo Kante were ruled out of the tournament by injury before Deschamps named his squad.
After opening their campaign against Australia, they will then play Tunisia and Denmark in Group D.
Off the pitch, FIFA has said it expects to generate 7.5 billion US dollars - roughly €7.25bn - in the four-year cycle up to the World Cup finals in Qatar.
The figure has been earned primarily from commercial deals – such as the one with Budweiser, who were blocked from selling beer at World Cup stadiums this week – and broadcasting.
The eye-watering sums again underline the commercial value of the World Cup to businesses and broadcasters, and the importance of the competition to FIFA itself.
The figures, which are up by around €970 million compared to the previous cycle, were relayed to national associations at a summit in Doha on the opening day of the World Cup.
FIFA president Gianni Infantino hits out at European criticism of Qatar
They will help to solidify political support for FIFA president Gianni Infantino, who is set to be re-elected unopposed next year.
FIFA sources say €5.2bn goes to cover football activities – including the forward programme which supports national associations and continental confederations and other talent development programmes.
It also covers the other competitions FIFA operates which run at a loss – the Club World Cup, youth tournaments and the 2019 Women's World Cup.
That has yielded a net profit of around €970m, with around €290m set aside to support federations through the coronavirus pandemic.
The profit is higher than the previous four-year cycle, owing to reduced staging costs for FIFA and higher sponsorship revenue, including from Qatari businesses.
Additional reporting: PA
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