The prolactin gene family in rodents consists of multiple members that coordinate the processes of reproduction and pregnancy. Some members of this family acquired one or two additional exons between exon 2 and exon 3 of the prototypical 5-exon, 4-intron structure, but the evolutionary importance of this insertion is unclear. Here, we focus on those members and survey this question by molecular evolutionary methods. Phylogenetic analysis shows that those members cluster into two distinct groups. Further analysis shows that the two groups of genes originated before the divergence of mouse and rat but after that of rodents from other mammals. We compared the d (N)/d (S) values for each branch of the gene tree but found no evidence to support positive selection for any branch. We found strong evidence, however, that one site (11E) of the 13 sites of the first extra exon underwent positive selection by the site-specific models of the maximum-likelihood method. Combining our molecular evolutionary analysis with other known functional evidence, we believe that the insertion of the extra exon implies some functional adaptation.