Abstract: The Myf-5 gene of the gayal (Bos frontalis) was cloned and sequenced by the T-A method, including the 5′flanking ragion, three exons, two introns and partial 3′flanking region, then based on the nucleotide and amino acid sequences compared with some species (cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish) by DNAMAN, BioEdit and DnaSP, bio-softs, and also constructed the molecular phylogenetic tree on nucleoside acid of the coding region of Myf-5 gene with method of NJ, ME and UPGMA. The results show that: ①The numbers of exons and introns of Myf-5 gene of gayal are the same with that of cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish, consisted by 3 exons and 2 introns, but the size had some differences. The size of 3 exons and 2 introns was 660，76，1 226，852，407 bp,respectively. The junction sequence of exons and introns of gayal’s Myf-5 gene was accordance with gene composition of GT-AG rule. ②The homology rates of nucleotide sequences of the coding region of Myf-5 gene between the gayal and cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish were 99.0%，90.0%，90.5 %，90.5%，87.2%，84.8%，72.9%，64.3%，51.8%，respectively; while the homology rates of amino acid sequences were 99.6%，94.9%，94.6%，94.9%，89.9%，88.7%，81.6%，70.4%，56.6%，respectively. This result showed that the nucleotide sequence of the coding region and accordingly amino acid sequences of Myf-5 gene of gayal and cow, chimpanzee, rhesus monkey, human, dog, house mouse, Chinese softshell, chicken and zebrafish were quite conservative. ③The result was basically consistent with three methods which was NJ, ME, UPGMA constructed in molecular phylogenetic tree, the gayal was clustered together with cow firstly, chimpanzee, rhesus monkey and human clustered together, then together with each other, followed by other species. The clustering result was accordance with the results of mtDNA, other function genes and the zoological classification. This indicated that Myf-5 gene fit to construct molecular phylogenetic tree.