Heat stress is one of the most detrimental environment stresses for plants. Hydrogen peroxide (H₂O₂) is produced quickly in response to various stresses and likely plays a positive role in transmitting stress signal in organisms. This investigation addressed whether an exogenous H₂O₂ application would affect the heat response of turfgrasses and induce acclimation. Tall fescue (Festuca arundinacea cv. Barlexas) and perennial ryegrass (Lolium perenne cv. Accent), two important cool-season turfgrasses and forages, were sprayed with 10 mM H₂O₂ before they were treated with heat stress (38/30°C, day/night) and compared with plants maintained at control temperatures (26/15°C, day/night). Prior to the initiation of heat stress, H₂O₂ pretreatment increased the activities of guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathionedependent peroxidase (GPX) and the ascorbate and glutathione pool, and it decreased the GSH/GSSG ratio. During the heat stress process, pretreated plants from both grasses exhibited higher turfgrass quality and relative water content, and they experienced lower oxidative damage and H₂O₂ levels. Moreover, the activities of APX, GR, GPX and glutathione-S-transferase increased significantly in response to H₂O₂ pretreatment under heat stress. These results suggested that H₂O₂ most likely participated in the transduction of redox signaling and induced the antioxidative defense system, including various enzymatic and nonenzymatic H₂O₂ scavengers. The scavengers played important roles in improving the thermotolerance of tall fescue and perennial ryegrasses.