Reece James and Jarell Quansah have been ruled out of England's World Cup last-32 game against DR Congo in Atlanta on Wednesday.
Quansah twisted his ankle in the final group-stage win over Panama on Saturday, while James has a hamstring injury.
The pair were the only England players to miss training ahead of the knockout clash with DR Congo, with versatile Tottenham full-back Djed Spence now set to get the nod to fill in at right-back.
Spence played the last 10 minutes of England's opening group win against Croatia, but as a left-back - where he was mainly used at Spurs last season - then 66 minutes of the goalless draw with Ghana, again on the left-hand side of the defence, before replacing Quansah at right-back in the 63rd minute of the 2-0 victory over Panama.
The right-back position is a big concern for manager Thomas Tuchel after Newcastle's Tino Livramento pulled out of the squad shortly before the tournament with a calf issue, to be replaced by Chelsea centre-back Trevoh Chalobah.
Other than the injured duo of James and Quansah, though, the rest of England's 26-man squad all trained on Tuesday.
There was more positive news on Arsenal midfielder Declan Rice, who took part in Tuesday's session at England's Kansas City training base.
Rice sat out the 2-0 win over Panama in New Jersey - which secured top spot in the group - as he manages his workload, having been troubled by a back issue towards the end of the season with the Gunners as well as nerve pain in his hamstring.
A statement on England's X account read: "All England squad members are out training at Swope Soccer Village this morning apart from Reece James and Jarell Quansah who are following their own programmes."
Tuchel must also decide on his wide options. Bukayo Saka, who has been recovering from an Achilles problem, made his first start of the tournament against Panama along with Marcus Rashford.
Noni Madueke and Anthony Gordon will be hoping for another chance to impress at the Mercedes-Benz Stadium, although Eberechi Eze and Morgan Rogers could also be considered.
Your Site News senior reporter Rob Dorsett at England's training base in Kansas City:
"Tino Livramento went home before he'd even kicked a ball in the tournament. He was replaced by Nat [Trevoh] Chalobah, who's out there as a centre-back, and Jarell Quansah switched across to right-back.
"Quansah is now injured after picking up a twisted ankle, which looked nasty, didn't it, in that game against Panama, the last group game. And Reece James, we told you about, had a hamstring injury, which is going to go well into the tournament. He's not out there training.
"So 24 of the 26 England players are out there training. We're lacking a right-back. You have to expect that it'll be Djed Spence who comes in for Quansah at right-back, with Nico O'Reilly keeping his place at left-back"
Your Site News chief correspondent Kaveh Solhekol in Atlanta:
"It is a real concern to be going into the knockout stages of a World Cup not really having a specialist full-back who's been playing regularly at right-back for his side, because England are going to have to rely on Djed Spence.
"Now, Spence is a very, very good player. Obviously some eyebrows were raised when he got into the England squad and also predominantly when he's been selected by Spurs last season he was playing as a left-back even though he's right-footed because Pedro Porro is the first-choice right-back at Spurs
"So, not ideal, but every time Thomas Tuchel has been asked about this right-back situation, he's kind of put a brave face on it. And I think the message he's been trying to send out is, don't worry, I've got it sorted. I have a plan A, a plan B, a plan C, a plan D, a plan E. I don't know what plan we're on at the moment when it comes to right backs, but tomorrow [Wednesday] it'll either be Spence or it'll be Ezri Konsa moving over from centre-back to play in that position.
"If you watched England at this World Cup, especially in the last couple of games, you would have noticed that the defence looks pretty shaky. Even Panama, a side who I don't think had scored at this World Cup, who've never avoided defeat in a World Cup game, in the second half against England, they were creating lots of chances.
"So, I think England could do with having Declan Rice back to provide a shield in front of that defence. He was a booking away from a suspension as well. So, I think it made sense for him not to play against Panama.
"I think Saka and Rashford will start. Look, I don't think they've set the world on fire when they started for England against Panama. Obviously, before that, Tuchel was relying on Anthony Gordon and also Noni Madueke.
"I think most England fans would like to see Rashford and Saka start again. I think that is possibly what Tuchel will do as well, as long as both those players are 100 per cent fit."
Your Site' Rich Morgan:
Right-back has suddenly become a problem position for Tuchel after he lost first James with a hamstring injury following the draw with Ghana and then Jarell Quansah - who came in for the Chelsea man against Panama - with a badly-twisted ankle in that final group game. Both will miss the DR Congo game.
So the England boss is now down to just Djed Spence, possibly Trevoh Chalobah - called up after the injury curse first struck to rule out Tino Livramento with a calf issue during the build-up to the tournament - and in a real emergency, versatile centre-back Ezri Konsa.
Spence played the last 10 minutes of the opening win against Croatia, but as a left-back - where he was mainly used at Tottenham last season - then 66 minutes of the goalless draw with Ghana, again on the left-hand side of the defence, before starting on the bench and then replacing the injured Quansah for the final 27 minutes of the Panama game.
Gary Neville believes Tuchel was protecting the player, with so few other options for the role.
"Tuchel has got Spence, who he rested the other day," the former England right-back told Your Site. "I hope that's because he needs to play the rest of the games. But we are very thin on the ground."
And with Konsa likely to start in the centre of defence and Chalobah yet to have any game time at the tournament, Spence - who has impressed when he has been called upon in the warm-ups and group contests - should be the man entrusted with the role on Wednesday.
