The Manchester City right-back sustained an apparent hamstring issue in the first half of Saturday's 1-0 loss to Brazil, also plunging his participation into next weekend's Premier League showdown with Arsenal into doubt.
Southgate had no information on the seriousness of Walker's injury immediately after the weekend's defeat, although he did confirm that captain Harry Kane (ankle) would not be available for the visit of the Red Devils.
Cole Palmer and Jordan Henderson will also require assessment after failing to make the cut on Saturday, where Ollie Watkins got the nod up front in Kane's absence but failed to justify his selection.
As a result, Ivan Toney could oust his Aston Villa counterpart to lead the line on Tuesday, where Jarrod Bowen and Marcus Rashford are competent options to replace Phil Foden and Anthony Gordon out wide as well.
However, Jude Bellingham - the victim of some rough treatment from Brazil on Saturday - should reprise his advanced role, while Declan Rice is not likely to be in any danger of losing his spot in the middle either.
There is scope for one change in midfield, though, as debutant Kobbie Mainoo - who caught the eye with one particularly slick turn on Saturday - is a candidate to demote Conor Gallagher down to the bench.
Full-backs alterations could also see Ezri Konsa and Joe Gomez step in for the stricken Walker and Ben Chilwell, but John Stones and Harry Maguire ought to continue in front of undisputed number one Jordan Pickford.
England possible starting lineup:
Pickford; Konsa, Stones, Maguire, Gomez; Mainoo, Rice; Bowen, Bellingham, Rashford; Toney