Species inhabiting in different thermal niches must have acquired thermal sensitivity suitable for the respective niches. Functional changes of thermal sensors could directly influence thermal perception, thus may have played crucial roles in thermal adaptation. Here we compared thermal responses of two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) inhabiting different thermal niches. We first compared behavioral responses and found that X. laevis is much more sensitive to heat stimulation than X. tropicalis. Primary cultured sensory neurons also exhibited similar difference between the two species. Thus, we compared thermal responses of an ion channel TRPV1, which serves as a heat sensor, by electrophysiological experiments. Clear species difference in TRPV1 was observed with repeated heat stimulation. X. laevis TRPV1 exhibited almost full activity in the first heat stimulation and its responses gradually deceased with repeated heat stimulation (desensitization). On the other hand, X. tropicalis TRPV1 exhibited only a partial response in the first heat stimulation and its responses gradually increased (sensitization). In order to estimate the evolutionary trajectory of TRPV1 channel property, we then compared thermal responses of TRPV1 from three additional clawed frog species. TRPV1 from all three species exhibited desensitization property to repeated heat stimulation. To infer the ancestral states of TRPV1 channel properties, we reconstructed the TRPV1 ancestral proteins and examined their heat responses. Ancestral TRPV1 exhibited desensitization properties to repeated heat stimulation, indicating that the heat responses of TRPV1 changed from desensitization to sensitization in the lineages leading to X. tropicalis. Moreover, we identified three amino acid substitutions that are largely responsible for the species difference of TRPV1 heat responses. These results suggest that subtle amino acid substitutions can cause functional changes in thermal sensors and may have served as a driving force for the evolutionary changes in thermal perception.