Skeletal muscles are composed of two major muscle fibre types, glycolytic and oxidative, which can be differentiated using their mitochondrial content. Mitochondria are a major generator of reactive oxygen species, and muscles have adapted them to possess oxidative resistance to counteract the oxidative damage. The present study aims to clarify the oxidative tolerance of heat stress (HS) in different types of skeletal muscles of broiler chickens. Exposure of 3-week-old broiler chickens to HS conditions (34 °C, 12 h) resulted in significantly higher lipid peroxidation in Musculus pectoralis (Pec), which consists entirely of glycolytic muscle fibres (type IIB), than in thermoneutral (TN) birds. This increase did not occur in gastrocnemius (Gas) muscle, which has a lower proportion of type IIB fibres (65–80%). HS treatment resulted in significantly higher mitochondrial H2O2 production in Pec muscle but not in Gas muscle. In both muscles, HS treatment did not alter the gene expression levels of cytosolic antioxidative enzymes, superoxide dismutase (SOD) 1, catalase and glutathione peroxidase-4. In Pec muscle, there was no difference in SOD2 mRNA levels between TN and HS birds, while avian uncoupling protein (avUCP) was significantly down-regulated by HS treatment. Conversely, in the Gas muscle of HS birds, SOD2 mRNA level was significantly increased while avUCP mRNA level was unchanged. Based on this evidence, it is suggested that the glycolytic muscle (e.g., Gas muscle) in broiler chickens is more susceptible to HS-induced oxidative disturbance, in which avUCP and SOD2 may be involved.