Southgate trialled a new three-man defence as England lost 1-0 to Germany in Dortmund on Wednesday night, with the manager's side impressing until Lukas Podolski's sublime second-half winner.
A late change to the squad saw the injured Chris Smalling withdraw with an unspecified problem. Uncapped Ben Gibson was handed a first call up as his replacement.
The Three Lions currently lead Group F with three wins and a draw from their opening four matches, but failure to beat Edgaras Jankauskas's Lithuania this weekend could cost them top spot.
The teams will also pay tribute to the victims of Wednesday afternoon's terror attacks in London, with the FA confirming that the "bravery" of those affected will be honoured at Wembley.
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England host Lithuania at Wembley Stadium on Sunday, March 26, with kick-off scheduled for 5pm.
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Joe Hart has been named captain, with fellow stand-in skipper Gary Cahill suspended, for the World Cup qualifier with Southgate boasting a fully fit 23-man squad.
Jamie Vardy started up front against Germany in front of Marcus Rashford, and the Manchester United striker is expected to again start on the bench against Lithuania which Jermain Defoe tipped to start.
West Ham midfielder Michail Antonio and Old Trafford pair Phil Jones and Chris Smalling have been forced to withdraw from the England squad due to injury.
Southgate did not name replacements for the former two though Smalling's withdrawal through injury saw uncapped Ben Gibson called up on Friday.
Andy Carroll, Danny Welbeck and Theo Walcott were among the most notable absentees from Southgate’s 26-man squad, while captain Wayne Rooney also misses out.
Sunderland’s 34-year-old striker Jermain Defoe was the most notable beneficiary of the attacking trio’s absence, with Southampton duo Nathan Redmond and James Ward-Prowse and West Brom midfielder Jake Livermore among the surprise inclusions.
Southgate may be tempted to start with a three-man defence once again and frontload the wings and attack, though the absence of both Cahill and Jones and the withdrawal of Smalling may make a back-two a safer option.
Lithuania's job has been made harder with a number of key absences, as Edwin Girdvainis (injury), George Freidgeimas (suspended) and Vytautas Andriuskevicius all miss out.
England squad: Fraser Forster, Joe Hart, Tom Heaton, Ryan Bertrand, Nathaniel Clyne, Michael Keane, Luke Shaw, Ben Gibson, John Stones, Kyle Walker, Dele Alli, Ross Barkley, Eric Dier, Adam Lallana, Jesse Lingard, Jake Livermore, Alex Oxlade-Chamberlain, Nathan Redmond, Raheem Sterling, James Ward-Prowse, Jermain Defoe, Marcus Rashford, Jamie Vardy
England XI: Hart, Walker, Keane, Stones, Bertrand, Dier, Alli, Lallana, Oxlade-Chamberlain, Sterling, Defoe
Lithuania XI: Setkus, Borovik, Vaitkunas, Klimavicius, Slivka, Kuklys, Zulpa, Novikov, Valskis, Chernykh, Matulevicius
2018 WORLD CUP QUALIFYING: GROUP F (EUROPEAN)
England have won the only two meetings between the two nations - 4-0 (home) and 3-0 (away) in the Euro 2016 qualifiers back in 2015.
BETTING ODDS (via 888 Sport)
- England to win: 1/13
- Lithuania to win: 28/1
- Draw: 9/1
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Odds correct as of March 26 at 12pm.