High temperature stress is one of the major factors limiting the growth and development of cool-season grasses during hot summer in warm climatic regions. Changes in the membrane lipid peroxidation, antioxidant content, as well as ultrastructure of chloroplasts were investigated in leaves of two cool-season turfgrass species, Festuca arundinacea (moderately heat sensitive) and Lolium perenne (heat sensitive), exposed to three temperature levels (38, 42, 46℃) for 14 h respectively with a heat acclimation pretreatment at 30℃ for 3 d in a growth chamber. Relative water content (RWC), cell membrane thermostability, malondialdehyde (MDA) content, accumulation of hydrogen peroxide (H2O2) and production rate of superoxide (O•-2), ascorbate (AsA) and glutathione (GSH) contents were measured in leaves of the two turfgrass species. The results showed as follows: (1) Significant accumulation of H2O2 and high production rate of O•-2 were observed in leaves of the two turfgrass species at all tested temperature levels regardless of whether heat-acclimated or not, but the degree of accumulation and the production rate in heat-acclimated turfgrass were lower than in non heat-acclimated turfgrass. (2) The changes in contents of AsA and GSH in the two turfgrass species showed significantly the trend of decrease with increase of high temperature. The contents of AsA and GSH in leaves of heat-acclimated turfgrass were higher than in non heat-acclimated turfgrass at same temperature level. (3) The degree of damage in chloroplasts increased with increasing of temperature. Ultrastructure of chloroplasts in heat-acclimated leaves was damaged less by high temperature stress than those plants without heat acclimation. The findings suggested that it might be helpful to protect the integrality of chloroplasts from the impact of high temperature stress by the heat acclimation pretreatment that induced lower membrane lipid peroxidation, higher AsA and GSH contents in leaves of two turfgrass species, which might be also one of the mechanisms for adaptation of cool-season turfgrass to heat stress.