The 31-year-old forward, who has lost his place in Manchester United's first team, was also left out by England earlier this year for matches against Germany and Lithuania.
Southgate named a 25-man squad which includes Tottenham Hotspur right back Kieran Trippier for the first time.
Sunderland striker Jermain Defoe, who scored on his return to the international scene against Lithuania in March after a long absence, retains his place in Southgate's plans.
Rooney, who has scored a record 53 goals for his country, has not featured in the squad since he was photographed socialising at a wedding reception at the England team hotel after the home victory over Scotland in November.
He later apologised to Southgate and Football Association technical director Dan Ashworth, saying images taken of him were "inappropriate for someone in his position".
Rooney holds the outfield record of 119 caps and needs seven more to surpass goalkeeper Peter Shilton's England record total.
England are favourites to qualify for the 2018 World Cup finals having seized control of the group.
They lead the section with 13 points from five games, four more than second-placed Slovakia.
Scotland are fourth six points off the pace and struggling to stay in contention for a place in Russia next year.
"The Scotland game is one of football's great fixtures," Southgate said.
"We're in a place in the group where a win can really put us in a very strong position, that's what we want to achieve."
England face Scotland at Hampden Park on June 10 before playing France in Paris three days' later.
Squad:
Goalkeepers: Jack Butland (Stoke City), Fraser Forster (Southampton), Joe Hart (Manchester City), Tom Heaton (Burnley)
Defenders: Ryan Bertrand (Southampton), Gary Cahill (Chelsea), Nathaniel Clyne (Liverpool), Aaron Cresswell (West Ham United), Ben Gibson (Middlesbrough), Phil Jones (Manchester United), Chris Smalling (Manchester United), John Stones (Manchester City), Kieran Trippier (Tottenham Hotspur), Kyle Walker (Tottenham Hotspur)
Midfielders: Dele Alli (Tottenham Hotspur), Eric Dier (Tottenham Hotspur), Adam Lallana (Liverpool), Jesse Lingard (Manchester United), Jake Livermore (West Bromwich Albion), Alex Oxlade-Chamberlain (Arsenal), Raheem Sterling (Manchester City)
Strikers: Jermain Defoe (Sunderland), Harry Kane (Tottenham Hotspur), Marcus Rashford (Manchester United), Jamie Vardy (Leicester City).
(Reporting by Martyn Herman; Editing by Ken Ferris)
