The Argentina star had pulled up in discomfort after tangling with Atletico defender Diego Godin, and was taken off by Luis Enrique as a precaution having initially attempted to play on.
Barca have now announced that Messi is expected to be sidelined until the middle of next month, most likely until after the international break.
“Leo Messi has a groin strain in his right leg and he will be out for around 3 weeks,” the club posted via their official Twitter account.
Messi sustained a groin problem during international duty with Argentina and only played 30 minutes as a second-half substitute during the shock 2-1 loss to Alaves on September 10, as Luis Enrique attempted to give him some extra rest.
He is set to miss LaLiga matches against Sporting Gijon and Celta Vigo, as well as a Champions League trip to Borussia Monchengladbach, although Barca will hope to have him fit for the home match against Pep Guardiola’s Manchester City on October 19.
Barca took the lead against Atletico through Ivan Rakitic’s first-half header, but Angel Correa equalised in the second period, just two minutes after Messi’s withdrawal.