The City defender was forced off with a problem in Tuesday’s Champions League win over Young Boys with boss Pep Guardiola admitting it is “deep bad news”.
Stones only returned to action in October after a two-month absence with hamstring and hip problems but Southgate had no hesitation in starting him in the important qualifying win over Italy.
But with qualification for the summer’s tournament in Germany already secured, Southgate – who names his squad on Thursday – can afford to look at his back-up options.
Brighton captain Lewis Dunk and AC Milan’s Fikayo Tomori started together in last month’s friendly win over Australia but Crystal Palace’s Marc Guehi was the man Southgate opted to replace Stones for the final 27 minutes against Italy and has the most caps of the trio.
Injuries to Ben Chilwell and Luke Shaw meant Kieran Trippier filled in at left-back against Italy but Chelsea’s Levi Colwill started against Australia and could be another one to benefit from any experimentation.
Colwill’s club captain Reece James could be in for a recall having returned from injury while Chelsea team-mate Raheem Sterling will be hoping two goals and some improved form in his last five matches will earn him a call-up for the first time since the World Cup.
Those chances could be enhanced depending on the extent of the ankle injury sustained by James Maddison during Tottenham’s loss to Chelsea on Monday and how significant the hamstring problem which forced off Callum Wilson in Newcastle’s Champions League defeat against Borussia Dortmund.
But Arsenal forward Bukayo Saka should return after withdrawing from the squad in October.
Manchester City midfielder Kalvin Phillips’ 49 minutes of action since his international start against Italy is unlikely to count against him, but James Ward-Prowse is knocking on the door of a first call-up in 14 months after a number of good performances for West Ham.