Crystal Palace centre-back Guehi and Southampton right-back Walker-Peters start the friendly at Wembley, while Palace left-back Tyrick Mitchell should come off the bench for his debut.
Guehi starts in a back three alongside John Stones and Conor Coady.
Jordan Henderson and Conor Gallagher form a midfield two, with Phil Foden and Mason Mount supporting Harry Kane.
Emile Smith Rowe is not in the squad missing training on Friday, while Bukayo Saka has pulled out of the squad because of Covid.
Guehi was named in the original squad but Mitchell and Walker-Peters were late call-ups after the withdrawals of Trent Alexander-Arnold and Reece James.
Southgate said: “We will have debuts this week for sure. Great moments for those players and we will always want to put them in with experienced players.
“Training has been good. The players have enjoyed being back together with their England team-mates.
“It has given them a psychological refresh before they go back into another important period with their clubs and we are enjoying working with them again.”
England XI: Pickford, Walker-Peters, Guehi, Coady, Stones, Shaw, Gallagher, Henderson, Mount, Foden, Kane
Subs: Pope, Forster Maguire, White, Mings, Grealish, Rice, Bellingham, Watkins, Sterling, Ward-Prowse, Mitchell
Date and time: 5.30pm GMT on Saturday March 26, 2022
TV channel: Sky Sports