Neymar will miss the 2019 Copa America on home soil after spraining a ligament in his ankle during Brazil's 2-0 friendly win over Qatar in Brasilia.
The Paris St-Germain forward, 27, who was stripped of the Brazilian captaincy in May, limped off after 17 minutes.
Everton's Richarlison had put the hosts ahead after 16 minutes and Manchester City striker Gabriel Jesus added a second eight minutes later.
The 12-team Copa America tournament starts on Friday, 14 June.
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Neymar was taken to hospital for a scan after being helped off the pitch by members of the Brazil support staff, having appeared to twist his ankle.
Brazil coach Tite, who on Monday described the forward as "indispensable" but not "irreplaceable" in the team, initially believed the injury would not be "very serious".
And on Saturday, a PSG club doctor assessed Neymar's injury, which does not require surgery and ruled him out for four weeks.
Neymar, who joined PSG from Barcelona for a world record 222m euros in August 2017, missed three months between January and April earlier this year after breaking a metatarsal in his right foot.
He also missed three months following surgery on the same injury in 2018, returning just in time to play in the World Cup.
Paris St-Germain team-mate Dani Alves replaced the forward as national team captain - a role he held for eight months - following disciplinary issues that included a ban for lashing out at a fan.
The hosts are attempting to win South America's continental tournament for the first time since 2007.
Neymar's injury came on the day a woman appeared on Brazilian TV to go public with accusations that she was raped by the player - accusations he denies.