Much of the DNA in genomes is organized within gene families and hierarchies of gene superfamilies. DNA methylation is the main epigenetic event involved in gene silencing and genome stability. In the present study, we analyzed the DNA methylation status of the prolactin (PRL) superfamily to obtain insight into its tissue-specific expression and the evolution of its sequence diversity. The PRL superfamily in mice consists of two dozen members, which are expressed in a tissue-specific manner. The genes in this family have CpG-less sequences, and they are located within a 1-Mb region as a gene cluster on chromosome 13. We tentatively grouped the family into several gene clusters, depending on location and gene orientation. We found that all the members had tissue-dependent differentially methylated regions (T-DMRs) around the transcription start site. The T-DMRs are hypermethylated in nonexpressing tissues and hypomethylated in expressing cells, supporting the idea that the expression of the PRL superfamily genes is subject to epigenetic regulation. Interestingly, the DNA methylation patterns of T-DMRs are shared within a cluster, while the patterns are different among the clusters. Finally, we reconstituted the nucleotide sequences of T-DMRs by converting TpG to CpG based on the consideration of a possible conversion of 5-methylcytosine to thymine by spontaneous deamination during the evolutionary process. On the phylogenic tree, the reconstituted sequences were well matched with the DNA methylation pattern of T-DMR and orientation. Our study suggests that DNA methylation is involved in tissue-specific expression and sequence diversity during evolution.