It comes after the forward clashed with Joe Gomez during Manchester City's loss to Liverpool, Sterling's former club.
Southgate's squad convened at St George's Park on Monday following the latest round of Premier League fixtures, which culminated in Sunday's highly-anticipated clash of the table toppers.
The PA news agency understands Sterling and Gomez were again involved in an exchange after meeting up for England duty on Monday, leading the Football Association to release a statement later that day.
A statement read: "The FA can confirm Raheem Sterling will not be considered for Thursday's EURO 2020 qualifier against Montenegro as a result of a disturbance in a private team area at St. George's Park today. He will remain with the squad."
It is a remarkable situation and is sure to dominate the build-up to Thursday's Euro 2020 qualifier against Montenegro, which not only offers the chance to seal progress but is England's 1,000th international fixture.
England boss Southgate said: "We have taken the decision to not consider Raheem for the match against Montenegro on Thursday.
"One of the great challenges and strengths for us is that we've been able to separate club rivalries from the national team.
"Unfortunately the emotions of yesterday's game were still raw. My feeling is that the right thing for the team is the action we have taken.
"Now that the decision has been made with the agreement of the entire squad, it's important that we support the players and focus on Thursday night."
Sterling has been in exceptional form for England, scoring eight goals and providing five assists during Euro 2020 qualification. Gomez, who was introduced as an 87th-minute substitute on Sunday, won the last of his seven England senior caps in the Nations League third-place play-off against Switzerland in June.