Tibetan sheep are highly adaptable to cold and hypoxic environments, allowing them to thrive on the Qinghai‒Tibet Plateau. Although many studies have investigated the mechanisms underlying their cold tolerance and hypoxia adaptation, it is still unclear how Tibetan sheep cope with the challenges of heat stress (HS) when raised in southern China. In this study, Tibetan sheep and heat-tolerant breeds (Hu sheep and Huanghuai sheep) were selected, and we conducted physiological observations, biochemical measurements, and metabolomic analysis on serum from a nonheat stress (NH) group, where the temperature-humidity index (THI) was 59.13, and a heat-stressed group (THI = 82.78). The results revealed that HS significantly increased the rectal temperature, respiratory rate, and heart rate of Tibetan sheep, and these values were significantly greater than those in the heat-tolerant breeds. Serum antioxidant indices revealed that catalase activity, total antioxidant capacity, and total superoxide dismutase activity were significantly lower in Tibetan sheep than in Hu sheep under HS. Metabolomic analysis revealed that HS reduced the levels of serum antioxidant metabolites, such as creatine, ornithine, l-arginine, and guanosine, and increased the levels of the oxidative stress marker metabolite 3-nitrotyrosine in Tibetan sheep. Additionally, the ratio of serum antioxidant metabolites to 3-nitrotyrosine in Tibetan sheep was significantly lower than that in heat-tolerant breeds, and these ratios were correlated with physiological parameters. Therefore, these results suggest that Tibetan sheep are relatively sensitive to HS.