Heat sensation and tolerance are crucial for determining species’ survival and distribution range of small mammals. As a member of the transmembrane proteins, transient receptor potential vanniloid 1 (TRPV1) is involved in the sensation and thermoregulation of heat stimuli; however, the associations between animal’s heat sensitivity and TRPV1 in wild rodents are less studied. Here, we found that Mongolian gerbils (Meriones unguiculatus), a rodent species living in Mongolia grassland, showed an attenuated sensitivity to heat compared with sympatrically distributed mid-day gerbils (M. meridianus) based on a temperature preference test. To explain this phenotypical difference, we measured the TRPV1 mRNA expression of two gerbil species in the hypothalamus, brown adipose tissue, and liver, and no statistical difference was detected between two species. However, according to the bioinformatics analysis of TRPV1 gene, we identified two single amino acid mutations on two TRPV1 orthologs in these two species. Further Swiss-model analyses of two TRPV1 protein sequences indicated the disparate conformations at amino acid mutation sites. Additionally, we confirmed the haplotype diversity of TRPV1 in both species by expressing TRPV1 genes ectopicly inEscherichia colisystem. Taken together, our findings supplemented genetic cues to the association between the discrepancy of heat sensitivity and the functional differentiation of TRPV1 using two wild congener gerbils, promoting the comprehension of the evolutionary mechanisms of the TRPV1 gene for heat sensitivity in small mammals.