The centre-back sustained bruising to his knee in the final part of Wednesday's (AEDT) training session and will not be risked for the visit to the Czech Republic capital.
Umtiti, who has only played twice for Barca this season due to injuries, will now face a race to be fit for next Tuesday's LaLiga match at home to Real Valladolid.
Sergi Roberto is out as he continues his recovery from a knee problem, while Carles Alena and Carles Perez are also sidelined.
Ernesto Valverde's side is otherwise mostly at full strength, with youngsters Ansu Fati and Inaki Pena included alongside the senior stars in the 20-man group.
Barca is second in Group F with four points from two games, behind Borussia Dortmund on goal difference.