Alex Cuthbert has joined Dan Biggar and Liam Williams in withdrawing from the Wales side to face South Africa in Cardiff on Saturday (15:15 BST).
Tom Rogers will replace Cuthbert, who has a tight calf, in a third change from the side initially named for Wales’ final World Cup warm-up game.
Sam Costelow is in for fly-half Biggar, who has a back spasm.
Cai Evans wins his first cap having replaced full-back Williams, who has a tight hamstring.
Louis Rees-Zammit is named as a replacement.
Cuthbert had also missed the training camps in Switzerland and Turkey and Wales’ opening two warm-up matches through injury.
The starting Wales backline now only have 39 caps between them, with scrum-half Kieran Hardy having 17 of those.
Head coach Warren Gatland will name his 33-man World Cup squad on Monday, 21 August.
Wales stressed the withdrawals of Biggar and Williams were precautionary and that they are not World Cup doubts.
“They are not serious at all,” said Gatland. “If this was a World Cup semi-final, Dan Biggar would have been fine for the weekend.
“We are just taking a precautionary measure. There is no need for us to take a risk with him.
“Both of them [Williams and Biggar], if we pushed them hard, could play on Saturday, but the decision was made [that] there is no point taking a risk at this stage.
“It’s dealing with things that are going to be thrown at you. It’s using a bit of adversity as a positive.”
Hooker Dewi Lake is in contention to be fit for the World Cup opener against Fiji in Bordeaux on 10 September with back row Taine Plumtree, hooker Ryan Elias and lock Dafydd Jenkins also targeting that game.
Cuthbert, number eight Taulupe Faletau (calf) and fly-half Gareth Anscombe (thumb) are the only players from the 48-man squad who have not been able to play in any of the three warm-up games.
Lake was forced off the field after captaining the side in the 19-17 defeat against England at Twickenham last weekend.
Dragons hooker Bradley Roberts has been called in to train with Wales to provide cover amid the injuries to Lake and Elias, who suffered a hamstring injury in the 20-9 opening World Cup warm-up win against England in Cardiff in early August.
Roberts’ fellow Dragons hooker Elliot Dee will start against South Africa with Sam Parry on the replacements bench.
Gatland said it was a balancing act about how many players he could select with fitness issues.
“We have in the past gone to World Cups carrying players with niggles that are going to take two or three weeks to come right,” said Gatland.
“That may be the case but it’s just depending on what sort of risk do you potentially take.
“There are maybe one or two we can take with niggles, that’s probably the number you talk about.
“The important thing is when you can get back to training. If you look at the draw, you’ve got to go fully loaded for the first game, then looking at Portugal with a six-day turnaround there’ll be changes.
“Then you’ve got eight days to Australia where you’ll be a strong side again and then a 13-day turnaround where hopefully everyone gets an opportunity to recover from any bangs and bruises before you play Georgia.
“There’s less pressure in this tournament than there has been in the past where you’ve had four-day turnarounds when that’s had quite a significant impact on the squad.
“That’s a discussion we’ll have in terms of risk and how many we can carry.”
Gatland insisted Dragons flanker Taine Basham was fit to play against South Africa after recovering from a high tackle by England captain Owen Farrell.
Basham, 23, failed his head injury assessment (HIA) on the day but Gatland had written in his newspaper column the process had been flawed because there was a television on in the same room.
The WRU say Basham is fit to feature on the bench because no concussion was confirmed following completion of all three stages of World Rugby’s HIA protocol.
“He’s had a full week with us from Monday, he passed his HIA 2 and 3, so no problem at all,” said Gatland.
“He felt fine after the game. He felt fine when he was on the field. It was an independent doctor who had a look and said they wanted him to have a HIA.”
An independent judicial committee rescinded Farrell’s dismissal against Wales, clearing him to play a key role for England at the upcoming World Cup.
World Rugby have now appealed against the decision with another hearing set to be arranged.
Gatland would not be drawn on the Farrell controversy after initially criticising the England fly-half’s tackle technique in his newspaper column.
“I was forthright about the fact he’s got to get his technique a little bit better,” said Gatland.
“It’s something we talk with players about: if you don’t bend at the hips then you’re putting yourself in a situation that you can potentially get yellow carded or red carded.
“I’d like to think all coaches talk about tackle technique and getting those things right. And if you don’t get those things right, then you can potentially put yourselves in a position where you can be carded.
“Everyone wants clarity and certainty about all aspects of the game.”
Wales team to face South Africa: C Evans; Rogers, Grady, J Williams, Dyer; Costelow, Hardy; Domachowski, Dee, Assiratti, Carter, Rowlands, Lydiate, Morgan (capt), Wainwright.
Replacements: Parry, N Smith, H Thomas, Teddy Williams, Basham, Tomos Williams, Llewellyn, Rees-Zammit.
