The Manchester United forward was unable to join in with the rest of Gareth Southgate’s squad on Thursday and he was the only member of a 22-man party not to take part in the Three Lions’ latest warm-up session.
Rashford is now expected to sit out a meeting with the Czech Republic at Wembley on Friday.
An ankle problem picked up against Liverpool on February 24 is still causing the Red Devils star problems.
He was unable to come off after suffering that knock due to the fitness issues endured by others as United had no choice but to keep him on the field and risked making the injury worse.
Rashford was briefly rested during a meeting with Crystal Palace, but still stepped off the bench in that contest.
He has continued to play a prominent role for United over recent weeks, with a dramatic late penalty converted in a Champions League clash with Paris Saint-Germain to send the Red Devils into the quarter-finals.
Regular outings at club level may, however, have taken their toll.
If Rashford misses out against the Czechs, then he will join a lengthy list of absentees.
There has been no suggestion that Rashford will follow them out of the squad, but that situation will be reassessed if he plays no part on Friday.
With England due to be back in action against Montenegro on Monday, the suggestion is that Rashford will not travel unless he is fully fit.
For now, Southgate appears set to have to shuffle his pack.
That could mean the likes of Jadon Sancho and debut hopeful Callum Hudson-Odoi coming into his plans for a starting berth.
United, meanwhile, will be watching on from afar and hoping that Rashford is returned to them ready to step straight back into club football.
They have already seen Belgian striker Romelu Lukaku pick up a foot problem during the latest break in domestic duties.
Alexis Sanchez is also nursing knee ligament damage and Ole Gunnar Solskjaer could do without another injury headache.
England do not appear set to take any unnecessary risks, with the depth in their ranks allowing them to find suitable cover for Rashford as they prepare to start out down the road to next summer’s European Championship.