A total of three Micropterus salmoides populations ["YouLu No.3" (YL), Taiwan population (TW) and introduced population (M. salmoides salmoide)(BF)] with an average body mass of (15.61±1.25) g were used in this study to study the acute high-temperature tolerance of different populations and the effect of high-temperature on gill tissue and antioxidant enzyme activity. The water temperature was raised from 27 to 35, 36, 37, 38, 39 °C with a rate of 2 °C/h and maintained for 12 h. Each temperature group included three replicates and ten fish per repeat. Half-lethal temperature (LT₅₀) was predicted according to the mortality rate. The results showed that fish death in three populations beginning to occur at 36 °C, the LT₅₀ of YL was 37.47 °C, which was significantly higher than that of TW (37.15 °C) and BF (37.01 °C). By subjecting M. salmoides to high-temperature stress, and the gill tissue damage of M. salmoides at different temperatures (27, 35, 37 and 39 °C) was compared and analysed, with the increase of temperature, the separation of the epithelium of gill lamellae from the basement membrane increased, and the gill lamellae gradually became shorter and thicker, and the gill tissue damage was the most serious at 39 °C. The activities of antioxidant enzymes of M. salmoides at different temperatures (27, 35, 37and 39 °C) and different stress times (0, 3, 6 and 12 h) at 37.5 °C showed that the activities of superoxide dismutase (SOD) and catalase (CAT) increased and then decreased with increasing temperature, and the highest activities were observed at 37 °C. The activities of SOD and CAT increased and then decreased with increasing stress time. SOD and CAT activities showed trends of increasing and then decreasing with increasing stress time, and the highest activity was observed at 3 h. In summary, the heat tolerance of YL was significantly higher than that of the TW and BF. The high temperature stress caused stress damage to M. salmoides. Our study provides basic data and theoretical references for breeding high temperature tolerance M. salmoides.