Officials announced the 185-pairing on Thursday. The UFC 224 pay-per-view event will take place inside Jeunesse Arena.
The announcement comes one week after Gastelum (14-3) told ESPN he was open to facing Souza in hostile territory. Souza, 38, recently moved to Orlando, but is originally from Manaus, Brazil.
Souza (25-1) is coming off a first-round knockout against Derek Brunson at UFC Fight Night on Jan. 27. The 39-year-old missed the second half of 2017 due to multiple injuries. He has never fought for a UFC title, despite an impressive 8-2 mark.
Gastelum, of Arizona, is also coming off a first-round stoppage. He knocked out former champion Michael Bisping in November.
Gastelum has expressed concern in the past about fighting in Brazil. The Brazilian athletic commission fined and suspended Gastelum in 2017, after he tested positive for traces of marijuana.
In a recent statement to ESPN, however, Gastelum said he would reconsider fighting there, if a UFC title shot was on the line.
“I would love to fight in Rio, and obviously a fight with Jacare would propel me to where I want to go,” Gastelum said. “I’m scared of that commission. I don’t know what to think of the commission.
“I should probably talk to someone in the commission or [UFC vice president of regulatory affairs] Marc Ratner.”