Trent Alexander-Arnold has been ruled out of Euro 2020 with a thigh injury picked during their friendly match against Austria at Wembley.
The Liverpool right-back hurt himself in the closing stages of Wednesday's friendly and a scan revealed he has suffered a quad tear.
The 22-year-old is expected to be sidelined for around four to six weeks and will return to his club for rehabilitation.
England Manager Gareth Southgate will not name a replacement until after Sunday's final warm-up game with Romania.
Alexander-Arnold's inclusion as one of four right-backs was one of the main talking points when Southgate named his squad last week, with the Liverpool player having missed the previous seven internationals.
England's first game at Euro 2020 is against 2018 World Cup finalists Croatia at Wembley on Sunday, 13 June.