The 51-year-old former Crystal Palace and Aston Villa defender has called up AC Milan defender Fikayo Tomori, Aston Villa striker Ollie Watkins and Arsenal goalkeeper Aaron Ramsdale.
Tomori has only made one senior appearance but has impressed in Italy since moving from Chelsea while Watkins has been called up after missing out on the final cut for the Euro 2020.
Manchester United striker Mason Greenwood misses out, while there is also no place for Burnley goalkeeper Nick Pope, Borussia Dortmund midfielder Jude Bellingham or Leeds forward Patrick Bamford.
Speaking about his decision to include Tomori, Southgate said:
"I thought he finished last season well.
"He's playing at a club where there's an expectation to win every week, so he's performing under pressure.
"I like Fikayo in terms of his aggressiveness as a defender.
"Because he's got speed, he's confident enough to defend on the front foot and his use of the ball is improving all the time."
And he also spoke about his reasoning for opting for Ramsdale over Pope.
He said: "Aaron was with us all summer and we had the opportunity to work with him for a long time and get to understand his strengths.
"Nick (Pope) is still I think coming back from injury a little bit, but I think Aaron is just in a better vein of form at the moment.
"From Aaron's perspective, the move to a bigger club with higher expectations and a style of play where he is asked to build with his feet from the back, he's coping really well.
"I think that's an important factor for us when looking at the biggest matches, having a goalkeeper who has really adapted well to a move and is full of confidence."