In order to explore the evolutionary differences between PIN3 genes in different species of Brassicaceae, the Brassica database and the Brassicaceae gene database were used. In this paper 18 PIN3 genes were identified by sequences blast in the database of 17 sequenced Brassicaceae species (there were two homologous PIN3 in Brassica napus). The phylogenetics of the genes and their predicted proteins (PIN3) were carried out by bioinformatics analysis. The results indicated that the similarity of 18 PIN3 gene sequences in the Brassicaceae species was 86.74%, while the proteins similarity was 90.93%.The PI of PIN3 protein was form 7.15 to 9.44, the PI of Brassica rapa was higher than those of others. The amino acid difference was found in several glycosylation sites of PIN3 protein with species diversity, which conformed to the amino acid substitution phenomenon and may result in difference function and activity of the protein in evolutionary relationships.