Imidazole dipeptides possess important bioregulatory properties in animals. This study aimed to evaluate the effect of high ambient temperature on muscle imidazole dipeptides (carnosine, anserine, and balenine) in broiler chickens. Sixteen 14-day-old male broiler chickens were divided into two groups, which were reared under thermoneutral (25 ± 1 °C) or cyclic high ambient temperature (35 ± 1 °C for 8 h/day) for 4 weeks. Chickens exposed to cyclic high ambient temperatures displayed lower skeletal muscle anserine and carnosine content than control chickens. Balenine could not be detected in the pectoral muscle of either group. The pectoral muscles of broiler chickens kept under cyclic high-temperature exhibited significantly lower mRNA expression of carnosine synthase 1, which synthesizes carnosine and anserine; but a significantly higher mRNA expression of carnosinase 2, which degrades carnosine and anserine. Our results suggest that heat exposure decreases pectoral imidazole dipeptide content in broiler chickens. This may be attributed to a lower expression of imidazole dipeptide-synthesizing genes, but higher levels of genes involved in their degradation.