Everton midfielder Barkley, 19, has impressed for the Toffees in recent months, scoring his first goal for the club in the 2-2 draw with Norwich two weeks ago.
Tottenham winger Townsend, 22, missed out on the European U21 Championships after being suspended for betting offences, but he has been in fine form both for Spurs and QPR, where he spent last season on loan.
Meanwhile, Liverpool forward Daniel Sturridge returns to the squad after missing the last selection through injury, with Celtic goalkeeper Fraser Forster replacing the injured Ben Foster.
Southampton forward Rickie Lambert, who scored on his England debut against Scotland earlier this summer, remains in the squad.
Raheem Sterling has been promoted from the U21 squad even though he has only played 18 minutes for Liverpool this season - club team-mate Jordan Henderson is left out, despite playing in every minute of both league matches so far.
There is no room for Manchester City defender Joleon Lescott, with Spurs counterpart Michael Dawson also absent.
The match against Moldova is at Wembley on September 6, with England travelling to Kiev to face Ukraine four days later.
England squad for World Cup qualifiers against Moldova and Ukraine:
Goalkeepers: Joe Hart (Manchester City), John Ruddy (Norwich City), Fraser Forster (Celtic)
Defenders: Phil Jones (Manchester United), Gary Cahill (Chelsea), Glen Johnson (Liverpool), Leighton Baines (Everton), Ashley Cole (Chelsea), Phil Jagielka (Everton), Chris Smalling (Manchester United), Kyle Walker (Tottenham Hotspur)
Midfielders: Steven Gerrard (Liverpool), Frank Lampard (Chelsea), Jack Wilshere (Arsenal), Michael Carrick (Manchester United), James Milner (Manchester City), Ashley Young (Manchester United), Tom Cleverley (Manchester United), Raheem Sterling (Liverpool), Ross Barkley (Everton), Andros Townsend (Tottenham Hotspur)
Forwards: Wayne Rooney (Manchester United), Daniel Sturridge (Liverpool), Danny Welbeck (Manchester United), Theo Walcott (Arsenal), Jermain Defoe (Tottenham Hotspur), Rickie Lambert (Southampton)