Wales will be without Gareth Bale for October's friendly against England and their Nations League matches against the Republic of Ireland and Bulgaria.
The forward, 31, was carrying a knee injury before rejoining Tottenham from Real Madrid on loan earlier this month.
"It's nothing serious but it's just one to keep an eye on," said Wales manager Ryan Giggs.
There is better news for Wales with the return of Juventus midfielder Aaron Ramsey to the squad.
Defender Rhys Norrington-Davies, on loan at Luton Town from Sheffield United, earns a first call-up.
The other uncapped player in the squad is Nottingham Forest's 19-year-old attacking midfielder Brennan Johnson, who is on loan at Lincoln City and was called up as cover for September's matches against Finland and Bulgaria.
In central defence, Bournemouth's Chris Mepham and Joe Rodon of Swansea City return after injury, but Luton's Tom Lockyer and Anderlecht's James Lawrence are left out.
Captain Ashley Williams, who is without a club, is also omitted but full-back and record cap holder Chris Gunter is included despite also being unattached.
Wales face England in a friendly at Wembley on Thursday, 8 October, before turning their attention back to the Nations League, taking on Republic of Ireland in Dublin the following Sunday and Bulgaria in Sofia on Wednesday, 14 October.
'Not a big injury' for Bale
Bale has been beset with various injuries during recent seasons but Wales manager Giggs played down the significance of the knee issue, which will rule him out of the next three matches.
"Unfortunately since our last camp Gareth has not trained. He's only just started getting on the grass the last few days and we've had conversations with both Gareth and the medical team and he's not quite right for this camp," said Giggs.
"It was picked up in the first game [against Finland on 3 September] and then he played the second game and it was fine, but after the 90 minutes against Bulgaria he just felt it a little bit.
"It has just taken a bit of time to settle down and he's not quite right.
"It wasn't a big injury and of course Gareth always keeps himself fit, so he's able to come back as soon as the injury is right. He will back pretty soon I would imagine."
Bale has returned to Tottenham on a season-long loan from Spanish giants Real Madrid, where he had spent seven years after signing from Spurs for a then world record £85m in 2013.
Despite scoring more than 100 goals and winning four Champions Leagues with Real, Bale found himself marginalised during the past two seasons and became eager to leave in order to secure regular first-team football.
"With him going to a new club, we had to think seriously about not only the short term but the long term, get Gareth back fit, get him playing and I look forward to seeing him in the Premier League," Giggs added.
"I'm happy for Gareth because he's at a club he knows well. He will be playing regularly and I'm happy Gareth is back at a club he knows so well."
Goalkeepers: Wayne Hennessey, Danny Ward, Adam Davies.
Defenders: Chris Gunter, Ben Davies, Connor Roberts, Ethan Ampadu, Chris Mepham, Joe Rodon, Neco Williams, Ben Cabango, Rhys Norrington-Davies.
Midfielders: Aaron Ramsey, Jonathan Williams, Harry Wilson, David Brooks, Joe Morrell, Will Vaulks, Matthew Smith, Dylan Levitt, Brennan Johnson.
Forwards: Hal Robson-Kanu, Daniel James, Kieffer Moore, Tyler Roberts, Ben Woodburn, Rabbi Matondo.