Liverpool midfielder Adam Lallana pulled up injured in his first training session since being re-called for England, and will now miss the upcoming matches against Spain and Switzerland.
Lallana, 30, missed out on a place in England's World Cup squad due to his ongoing injury problems; having made just 13 appearances in all competitions for Liverpool last season.
Since returning to fitness, Lallana has put in a solid pre-season with Jurgen Klopp's side and has once again caught the eye of the England boss ahead of the first international break of the campaign.
However, a groin injury has now forced the midfielder, who has 34 caps to his name, out of the Three Lions squad and back to his club's Melwood training base for further assessment.
News broke on Monday that Raheem Sterling would miss the Nations League opener against Luis Enrique's Spain and, while Southgate was planning on not calling up any further players to replace Sterling in the squad, Lallana's injury may force the England boss to reconsider.
Southgate is not due to speak to the media until Friday, giving him some time to consider his options - and with Saturday's match being played at Wembley, any potential replacements will not have far to go to join up with the England squad.
Liverpool players on England duty historically have a bad record with injuries, with Daniel Sturridge and Joe Gomez both recent examples of Reds players who have picked up new fitness issues while on international duty.