AbstractRNase5 is an important member of the RNASE A superfamily. It has been considered as an intriguing model in molecular evolution studies. 17 species belong to 10 families from three evolution branches of Rodentia are analysised. RNase5 is detected in each genome by TBlastN and BlastN，which suggest the gene is losted in all species of Ctenohystrica after the evolution clade formed. Reconstructed RNase5 trees based on both Neighbor-Joining and Maximum likelihood methods supported three independent gene duplications are observed in Mus musculus, Rattus norvegicus and Peromyscus maniculatus in the mouse-related clade suborder. Strong positive selection was detected in the branch model，site model and branch-site model of PAML. In sum, the present work demonstrated the diversity about RNase5 studies, that provides an important foundation for further studies adaptive evolution mechanism of the RNase5.