Toll-like receptors (TLRs) are a family of proteins that recognize highly conserved pathogen- related molecular patterns (PAMPs) in pathogens, and play an important role in initiating innate immunity. Point mutations in the human tlr4 gene have been shown to be associated with many diseases, such as respiratory syncytial virus infection, atherosclerosis, malaria, etc., and these mutations are often racially specific. The Chinese Crocodile Lizard (Shinisaurus crocodilurus) is an ancient reptile which belongs to the national first-class protection animal. Due to anthropogenic disturbance, such as illegal hunting and habitat destruction, the number of the Chinese Crocodile Lizard is extremely rare. In recent years, a large number of death of the Chinese Crocodile Lizard has been caused due to various diseases, and the diseases are different among different populations, often with different disease symptoms, which are possibly caused by different pathogens. We predict that point mutations in the tlr4 gene might be distributed differently among the Chinese Crocodile Lizard populations to accommodate different local pathogens. Therefore, the aim of this study is to preliminarily explore the genetic diversity of tlr4 gene and the distribution characteristics of SNPs in the Chinese Crocodile Lizard by using PCR product direct sequencing and bioinformatics analysis, and to verify whether the point mutation on the gene has the population-specific distribution, and to test whether its non-synonymous substitution has an impact on the structure and function of protein. The amplified fragment length was 1 694 bp (Fig. 1), and the genetic diversity of each population was different, with Linzhouding population the highest and Dayaoshan population the lowest, which corresponds to the largest population of Linzhouding and the smallest population of Dayaoshan (Table 1). A total of 27 point mutations were found in 52 individuals (GenBank accession: MN380726﹣MN380777) from five populations, leading to nine non-synonymous substitutions (Table 2), three of which were predicted to affect the function and structure of proteins (Table 4). Twelve of the 27 point mutations were distributed in only one population, while four point mutations were not specific to any population, with wild-types and mutant types in each population, and the other eleven point mutations were distributed in two to three populations (Table 3). Only three of the 31 haplotypes were shared among populations, while the rest were distributed in a single population (Fig. 2). The results have shown that the genetic polymorphism of tlr4 gene is not consistent among different populations, and point mutations have population-specific distribution characteristics, reflecting the fact that different geographical populations are affected by different pathogenic pressures. It is suggested that attention should be paid to the maintenance of genetic diversity in future conservation of this species.