The cDNA sequence of bovine prochymosin gene was cloned and sequenced from the abomasums of suckling calf by RT-PCR. The sequence was aligned and bioinformatically analyzed with related sequences in GenBank. The result of sequence analysis revealed that the gene was determined to bovine prochymosin B gene and had the high level of homology with prochymosin gene of other known mammals. The base bias of 18 species of prochymosin gene reduced according to codon position, and the gene provided us with excellent material ofphylogenetic research. Thus, the phylogenetic tree of 18 species of prochymosin gene was used to discuss and offer testimony to phylogenetic relationship of 11 mammals.