Alex Scott and Ezri Konsa have pulled out of England's squad to face Czechia.
Scott has a thigh injury and is set for several weeks on the sidelines, sources told ESPN.
Bournemouth's Scott and Arsenal's Konsa have returned to their clubs for treatment.
Thomas Tuchel's depleted England squad will include Rico Lewis, who has been added to bolster the numbers after helping the Under-21s qualify for the 2027 UEFA U21 Euros. Manchester City's Lewis is seeking a sixth full international cap.
Scott made his full international debut in England's 7-0 victory over Croatia. Konsa also started.
Tuchel's other midfield options against Czechia in Scott's absence include Elliot Anderson, Jude Bellingham, Myles Lewis-Skelly, Morgan Gibbs-White, Eberechi Eze and James Garner. Trent Alexander-Arnold, who has operated in midfield, is also available.
Centre-back options include Marc Guéhi, Trevoh Chalobah and Jarrad Branthwaite. Bournemouth, without their star midfielder Scott, return to Premier League action against Chelsea this weekend. Arsenal will host Leeds United.
Information from ESPN's James Olley contributed to this report.