Heat stress affects a broad spectrum of cellular components andmetabolism.The objectives of this study were to investigate the behavior of Photosystem II (PSII) in tall fescue (Festuca arundinacea Schreb) with various thermotolerance capacities and to broaden our comprehension about the relationship between thermotolerance and PSII function. Heat-tolerant and heat-sensitive accessions were incubated at 24 C (control) and 46 C (heat stress) for 5 h. The fluorescence transient curves (OJIP curves), slow Chl fluorescence kinetic, and light response curve were employed to study the behavior of PSII subjected to heat stress. After heat stress, performance index for energy conservation from photons absorbed by PSII antenna until the reduction of PSI acceptors (PITotal), the value of electrons produced per photon (a), and the maximal rate of electron transport (ETRmax) of heat-tolerant accessions were lower than those of heat-sensitive accessions. Relatively lower reactive oxygen species (ROS) contents were detected in heat-tolerant accessions. Simultaneously, therewas a significant decline in the quantum yield of photochemical energy conversion in PS II (Y(II)), probability that a PSII Chl molecule functions as reaction center (cRC), and the increase of quantum yield for non-regulatednon-photochemical energy loss (Y(NO)) in heat-tolerant accessions. Moreover, a significant inverse correlation between heat tolerance indexes (HTI) andY(II)was observed. Therefore,maintaining a lower photochemical activity in heattolerant accessions could be a crucial strategy to improve their thermotolerance. This finding could be attributed to the structural difference in the reaction center, and for heat-tolerant accessions, it could simultaneously limit energy input into linear electron transport, and dissipate more energy through non-regulated non-photochemical energy loss processes.