Wolves' progression was secured on Friday night as they defeated National League North side Chorley 1-0.
The two sides played out a 1-1 draw in the West Midlands in November, with Theo Walcott's opener cancelled out by Pedro Neto's close-range strike.
The tie will be played at Molineux in the week commencing February 8.
Their run so far
Both sides have beaten a Premier League side and a lower league side to get to the fifth round.
Wolves beat Crystal Palace 1-0 in their third round tie a fortnight ago, but were lucky to avoid humiliation in the fourth round as Vitinha's wonder strike saw Wanderers past a dominant Chorley side.
Southampton have won two FA Cup ties in five days, as goals from Dan N'Lundulu and James Ward-Prowse saw off Shrewsbury in their rearranged tie before Gabriel's own goal was enough for the Saints to beat holders Arsenal.
Previous cup meetings
The sides have met in four FA Cup meetings before, although three of those came before World War I.
The most recent encounter came in the 2003 quarter-finals at St Mary's, as Premiership Southampton beat First Division Wolves 2-0 on their way to reaching the final in Cardiff, where they lost to Arsenal.
However, the sides did meet in the Carabao Cup in 2017, as goals from Danny Batth and Donovan Wilson saw second tier Wolves beat their top-flight opponents on the South Coast.