Dropped for World Cup qualifiers in March, the 22-year-old Liverpool player had won a recall amid much speculation as one of four right backs in Southgate's provisional 33-man squad and then the final 26.
His first match back ended in obvious pain after some 88 minutes at Middlesbrough's Riverside stadium when he signalled he had pulled a muscle while kicking the ball with nobody near him.
With England having used up all their substitutes he lingered on the sidelines and was then booked for returning to the pitch without permission.
Medical staff helped him back to the bench as he covered his face with his shirt. ITV television speculated he had mouthed 'it's gone'.
"It's not a good sign to see him have to walk off as he did," said Southgate.
"We'll know more in the next 24-48 hours."
Former Ireland international Roy Keane, in the ITV studio as a pundit, agreed the signs looked ominous.
"He was walking past us there and he looked really uncomfortable," said the ex-Manchester United captain. "He was swearing quite a bit so it doesn't look good. Absolutely not. The guy looks in big trouble."
Former England striker Ian Wright also sounded pessimistic.
"You could see, even when he was covering his face with his shirt, he himself probably feels it's not going to happen for him," he said.
"You know instantly when you are injured and how long it's going to be. It looked like thigh or hamstring... if it's that, and we're talking about 11 days, there's no way he's going to be able to make that." (Reporting by Alan Baldwin in London, editing by Toby Davis)
