In order to explore the molecular characteristics and evolution of the miR159 family members, we studied the miR159 family members registered in the miRBase database by distribution statistics, constructing the phylogenetic trees, comparing different families, and predicting their secondary structures and target genes. The results showed that the miR159 family was widely distributed in the plant community, and the ferns may be the evolutionary ancestors of the miR159 family. Phylogenetic tree analysis showed that there were multiple evolutionary directions among the members of the plant miR159 family, but the miR159 family members of the same type of plants tended to be clustered, and the members from the same evolutionary direction were highly homologous. Mfold predicts that premiR159 spontaneously forms a typical, stable stemloop secondary structure, and contains functional fragments ranged from 19~21 bases, which may be miR159 mature body. Target gene analysis showed that miR159 family members mainly regulate MYB transcription factor, transposable factor and hypothetical protein, but the types and numbers of target genes were differences among different species and among different members of the same species, especially between miR1593p and miR1595p.