The 33-year-old former Real Madrid star has broken his leg in a friendly against Malaga on Saturday.
The club indicated that the injury is a "new non-displaced fracture to the middle third of his left fibula"
Isco, who lifted the Champions League trophy four times during his time at Real Madrid, experienced the same injury in May 2024.
He made his return last December and assisted Betis in finishing sixth in La Liga, securing a spot in the Europa League, and captaining them in the Europa Conference League final, where they were defeated 4-1 by Chelsea
His performance led to a call-up to the Spanish national team for the first time since 2019.
He came off the bench and scored during the penalty shootout defeat to Portugal in the Nations League final.