Midfielder Barkley, 19, has featured in both of Everton's matches so far this season, scoring once against Norwich City.
Meanwhile, 22-year-old winger Townsend started for Tottenham Hotspur last time out against Swansea City. He also featured on a regular basis while on loan at Queens Park Rangers last term.
Elsewhere, Southampton striker Rickie Lambert has retained his place following his goalscoring debut against Scotland earlier this month, while Celtic goalkeeper Fraser Forster has been selected to replace the injured Ben Foster.
Liverpool's Raheem Sterling has also been included within the 25-man squad.
England squad to face Moldova and Ukraine:
Goalkeepers: Hart, Forester, Ruddy
Defenders: Baines, Cahill, Cole, Jagielka, Johnson, Jones, Smalling, Walker
Midfielders: Barkley, Carrick, Cleverley, Gerrard, Lampard, Milner, Sterling, Townsend, Walcott, Wilshere
Strikers: Defoe, Lambert, Rooney, Sturridge