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Abstract: The membrane lipid peroxidation, antioxidant metabolites, as well as ultrastructure of chloroplasts of leaves were investigated in two cool-season turfgrass species, perennial ryegrass (heat sensitive) and tall fescue (moderate), exposed to three heat stress levels (38, 42, 46°C) for 14h, respectively, with a heat acclimation pretreatment at 30°C for 3 days in a growth chamber. Relative water content (RWC), cell membrane thermostability, malondialdehyde (MDA) content, accumulation of hydrogen peroxide (H2O2) and superoxide (O2 −), ascorbate (AsA) and glutathione (GSH) contents were measured in leaves. RWC decreased with increase in heat stress levels in tall fescue and perennial ryegrass leaves. However, leaves with heat acclimation pretreatment kept lower decrease in RWC than those without heat acclimation pretreatment at three heat stress levels. Under heat stresses, leaves heat-acclimated pretreatment in two turfgrass species maintained higher membrane thermostability and lower membrane lipid peroxidation product (MDA) than those in no heat-acclimated leaves; however, perennial ryegrass leaves showed greater membrane injury than tall fescue leaves regardless of heat acclimation pretreatment, especially at 46°C. Significant accumulation of H2O2 and O2 − was observed in two turfgrass species leaves regardless of heat-acclimated pretreatment at all stress levels; however, their degree of accumulation kept lower level in heat-acclimated leaves than in no heat-acclimated leaves. AsA and GSH are two important metabolites in antioxidant system in plants. Our results showed the contents of AsA and GSH in heat-acclimated leaves were higher than those in control at same heat stress level though the contents of AsA and GSH showed the trend of significant decrease with increase in heat stress levels. After heat acclimation pretreatment, ultrastructure of chloroplasts had lower damage in two turfgrass species leaves under heat stress than those without heat acclimation pretreatment, which may contribute to the decrease in membrane lipid peroxidation, increase in AsA and GSH contents at same heat stress level. However, it was insufficient to protect the chloroplast from damage with increase in heat stress level, even if with high AsA and GSH contents to remove reactive oxygen species (ROS). [Copyright &y& Elsevier]