The Paris Saint-Germain star played the full 90 minutes and set up Lucas Paqueta's winning goal as Brazil beat Colombia on Thursday to book their spot at the 2022 tournament in Qatar.
But he will not face Argentina, who could also qualify with victory against Brazil on Tuesday.
"After training this morning, Neymar complained of thigh pain," the CBF said.
"Unable to carry out additional tests, the staff decided to rest the player who will not be travelling with the delegation to San Juan."
PSG will hope the 29-year-old Neymar is fit for their Champions League trip to Manchester City next week.
Neymar has scored 70 international goals, seven fewer than Brazil's all-time record scorer Pele.
Earlier on Monday, Argentina coach Lionel Scaloni said Neymar's club teammate Lionel Messi, who only played 15 minutes off the bench in Thursday's 1-0 win over Uruguay, will be fit to play in Tuesday's match.
Standout matches today