This study investigated the effects of long-term heat exposure on Japanese flounder (Paralichthys olivaceus) and its hybrids (P. olivaceus ♀ × summer flounder Paralichthys dentatus ♂). From 24 ± 0.5 °C, temperature was increased by 1 ± 0.5 °C in a day and was kept at that temperature for 5 days before next rise. Cumulative survival rate (CSR), cumulative survival rate under different temperature (CSR-T), histological alteration, and related enzyme activities were investigated. In P. olivaceus, mass mortality occurred at 29 and 32 °C (the CSR-T dropped to 42.39 %), and serious gill damages appeared at 30 and 32 °C. Meanwhile, the activities of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), and pyruvate kinase (PK) declined around 29 and 32 °C (except for CAT). In comparison with P. olivaceus, the CSR of the hybrids was higher, the gill kept a better structural integrity, and the activities of SOD, CAT, LZM, and PK showed tiny fluctuations. The results suggested that during the process of chronic heat stress, P. olivaceus seemed to be more sensitive to 29 and 32 °C, and the manifestations in survival, histology, and enzyme activity were generally consistent. For the hybrids, the comparatively insensitivity to high temperature might imply its better heat tolerance.