Smith Rowe made his senior international debut last November and scored his first England goal in the 10-0 World Cup qualifying win over San Marino, before keeping his place for the March international break.
However, he has started just one of Arsenal’s last five Premier League games and was overlooked by Southgate as West Ham’s Jarrod Bowen earned his first call-up.
Lee Carsley’s U21s face four European Championship qualifiers next month, away to Czech Republic and Kosovo and at home to Slovenia and Albania.
Smith Rowe’s Arsenal team-mate Folarin Balogun has also been included after spending the second half of the season on loan at Middlesbrough, as have Tottenham’s Ryan Sessegnon, West Ham full-back Ben Johnson and Hammers target Keane Lewis-Potter, of Hull.
The latter pair received their first call-ups in March alongside Nottingham Forest loanee Jed Spence, who will play in this Saturday’s Championship play-off final and has been linked with several Premier League clubs.
Everton starlet Anthony Gordon, Aston Villa’s Jacob Ramsey and Liverpool pair Harvey Elliott and Curtis Jones are among the top-flight regulars involved.
Latest England Under-21 squad
Goalkeepers: Joe Bursik (Stoke), James Trafford (Bolton), Carl Rushworth (Walsall)
Defenders: Max Aarons (Norwich), Charlie Cresswell (Leeds), Taylor Harwood-Bellis (Man City), Levi Colwill (Chelsea), James Hill (Bournemouth), Luke Thomas (Leicester), Ben Johnson (West Ham), Djed Spence (Middlesbrough)
Midfielders: Ryan Sessegnon (Tottenham), Tommy Doyle (Man City), Jimmy Garner (Man United), Morgan Gibbs-White (Wolves), Jacob Ramsey (Aston Villa), Curtis Jones (Liverpool), Harvey Elliott (Liverpool)
Forwards: Folarin Balogun (Arsenal), Anthony Gordon (Everton), Angel Gomes (Lille), Keane Lewis-Potter (Hull), Emile Smith Rowe (Arsenal), Cameron Archer (Aston Villa)