Toll-like receptor () genes that can recognize various pathogens are important innate immune members; thus, they are a hot spot in fish molecular immunology studies. The great blue-spotted mudskippers () inhabit an amphibious environment that has numerous pathogenic microorganisms. Based on this situation, it can be speculated that the might encounter strong selection pressure. In the present study, the complete sequence of and . nucleotide and amino acid sequences of were analyzed using molecular bioinformatics. The molecular evolution analysis of the 5 genes was conducted based on the phylogenetic tree. The tissue expression patterns of the 5 challenge were explored using real-time quantitative PCR. The results showed that the gene was 3071 bp and included a coding region that was 2646 bp, which encoded 882 amino acids. The gene was 3175 bp, and it had a 3033 bp coding region, which encoded 1011 amino acids, and the gene was 3398 bp and had a 3093 bp coding region, which encoded 1031 amino acids. The TLR5, TLR8, and TLR9 molecule structures were similar to those of other species, showing high conservation. The site model detected two sites (647Q, 691S) under positive selection pressure from fish 9 genes. Although there were no positive sites in the other 3 TLR3, and had some adaptive changes during long-term evolution. Furthermore, the branch-site model detected 3 positive sites (76P, 596A, and 603E) from the branch of The results of the branch-site model showed that owing to the transition from the aquatic environment to the amphibious environment, the underwent adaptive evolution. The results of expression patterns in healthy tissues showed that 5 genes were widely expressed in the tissues examined (intestine, eye, kidney, liver, brain, muscle, spleen, and skin), especially in the liver and spleen. Under genes in the liver and spleen increased at first and then decreased, suggesting that the play an important role in coping with bacterial invasion.