Climate change is causing frequent and intensely warm winters. As a result, ectothermic animals are expected to face more frequent periods of heat stress during winter dormancy. However, how heat exposure affects oxidative stress and antioxidative defense in hibernating reptiles remains largely unknown. In this study, we explored the effect of acute heat stress (25℃) on lipid peroxidation product malondialdehyde (MDA) and the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in hatchlings of Asian yellow pond turtle (Mauremys mutica) from high and low latitudinal populations during hibernation at 6℃. MDA content was higher in the livers of turtles from the southern population (P < 0.05), and increased further after turtles were returned to hibernation conditions (P < 0.05). MDA content in muscles of turtles from the northern population increased after heat stress (P < 0.05), but decreased to a level similar to that of the control group after moving back to 6℃ for 24 h (P > 0.05). SOD activity was not influenced by heat stress (P > 0.05). In livers of turtles from the southern population, CAT activity reached its highest level after heat stress for 2 h, and was significantly greater than that of turtles from the northern population at each sampling time (P < 0.05). GSH-Px activity in livers was greatest after heat exposure for 12 h in turtles from the northern population (P < 0.05). In conclusion, heat stress caused oxidative damage to M. mutica during hibernation. The southern population may be more vulnerable to temperature fluctuations during hibernation, but it also has stronger antioxidant enzyme activity in response to the threat of heat stress.