This is England's first squad since Wayne Rooney announced his international retirement.
In the first England squad since Wayne Rooney's international retirement, Southgate named Leicester defender Maguire and Watford midfielder Chalobah in a 28-man group to face Malta and Slovakia in World Cup qualifiers on September 1 and 4 respectively.
Southgate had asked Rooney to join the squad, but the Everton striker, who is England's all-time leading scorer, on Wednesday announced he has decided to call time on his international career at the age of 31.
Southgate is relishing the chance to see more of Maguire as injury prevented him being called up for England's World Cup qualifying draw against Scotland and friendly defeat by France at the end of last season.
Maguire is in his first season with Leicester after leaving Hull and Southgate said: "I thought his performances for Hull were very good. He's a defender that's very comfortable with the ball and that's the profile of international football.
"I think he's already adjusted really well at Leicester to a new club. He plays with great composure and can defend the ball in the box.
"So I think it's a really good opportunity for us to have a look at him."
Chalobah was allowed to leave Chelsea in the close-season after struggling to break into Antonio Conte's side, but Southgate has been impressed by his performances for Watford and England's Under-21s.
"Nathaniel has started the season extremely well with Watford. He's tactically an intelligent player and we know what he can bring," he said.
Arsenal forward Danny Welbeck returns to the squad having not appeared since a win over Germany in March 2016, but Liverpool midfielder Adam Lallana misses out due to injury.
Southgate is optimistic about the future after England won the Under-20 World Cup and Under-19 European titles, while other age group teams reached the European Under-21 and Under-17 Championship semi-finals.
"You can see by the squad we've named, some younger players coming in, who we've been looking at for a while and we want to see that. We have to have an eye on developing the future," he added.
"I did see the value of having some senior players around that. The likes of Jermain Defoe for example, who can help nurture some younger players as well as play a part - but equally we have to look forward."
Goalkeepers: Joe Hart (West Ham), Jack Butland (Stoke), Jordan Pickford (Everton), Tom Heaton (Burnley)
Defenders: Ryan Bertrand (Southampton), Aaron Cresswell (West Ham), Gary Cahill (Chelsea), Phil Jones (Man Utd), Harry Maguire (Leicester), John Stones (Man City), Michael Keane (Everton), Chris Smalling (Man Utd), Kyle Walker (Man City), Kieran Trippier (Tottenham)
Midfielders: Eric Dier (Tottenham), Nathaniel Chalobah (Watford), Jake Livermore (West Brom), Jordan Henderson (Liverpool), Raheem Sterling (Man City), Alex Oxlade-Chamberlain (Arsenal), Dele Alli (Tottenham), Jesse Lingard (Man Utd)
Forwards: Marcus Rashford (Man Utd), Danny Welbeck (Arsenal), Harry Kane (Tottenham), Jamie Vardy (Leicester), Daniel Sturridge (Liverpool), Jermaine Defoe (Bournemouth)