The under-21s face Kazakhstan on October 6 and Bosnia-Herzegovina five days later in Euro 2017 qualifying.
Chelsea striker Tammy Abraham, who is on loan at Bristol City, and Derby goalkeeper Jonathan Mitchell are called up for the first time.
England Under-20s manager Aidy Boothroyd will take charge of the under-21 team, with Gareth Southgate stepping up to lead the senior team for four matches after the departure of Sam Allardyce.
BBC Sport’s Simon Stone
Given Rashford scored a hat-trick for Gareth Southgate on his England Under-21 debut against Norway, it is not a surprise his name is missing from Aidy Boothroyd’s squad.
Former senior boss Sam Allardyce had been concerned at Rashford’s lack of football in August.
There are no such issues in September – he has played more matches (five) and minutes (316) for United this month than Wayne Rooney (four and 277 respectively).
Goalkeepers: Jonathan Mitchell (Derby County), Jordan Pickford (Sunderland), Freddie Woodman (Newcastle United)
Defenders: Calum Chambers (Middlesbrough, loan from Arsenal), Ben Chilwell (Leicester City), Brendan Galloway (West Bromwich Albion, loan from Everton), Rob Holding (Arsenal), Mason Holgate (Everton), Dominic Iorfa (Wolverhampton Wanderers), Matt Targett (Southampton)
Midfielders: Lewis Baker (Vitesse Arnhem, loan from Chelsea), Nathaniel Chalobah (Chelsea), Isaac Hayden (Newcastle United), Will Hughes (Derby County), Ruben Loftus-Cheek (Chelsea), John Swift (Reading), James Ward-Prowse (Southampton)
Forwards: Tammy Abraham (Bristol City, loan from Chelsea), Demarai Gray (Leicester City), Jack Grealish (Aston Villa), Nathan Redmond (Southampton), Duncan Watmore (Sunderland)