The Manchester United forward, who is struggling to overcome the ankle injury he sustained in last month’s Premier League clash with Liverpool, is a concern for the visit of the Czechs to Wembley.
Rashford has been eager to prove his fitness, undertaking an individual indoor session at St George’s Park on Thursday morning, and is keen not to withdraw from the England squad.
Nonetheless, manager Gareth Southgate may opt against risking the 21-year-old, whose absence is likely to pave the way for a Sancho start.
Rashford’s setback is another blow for Southgate who has already lost John Stones, Fabian Delph, Ruben Loftus-Cheek, Luke Shaw and Trent Alexander-Arnold to injury.
Those withdrawals have seen Southampton’s James Ward-Prowse and Chelsea’s Callum Hudson-Odoi earn late call-ups.