The dehydrogenase/reductase (SDR family) member 4 (DHRS4) gene is copied during mammalian evolution; therefore, while only one DHRS4 gene is expressed in the mouse genome, the gene cluster consists of two (DHRS4 and DHRS4L1) and three (DHRS4, DHRS4L2, and DHRS4L1) copies in chimpanzees and humans, respectively. In this study, we explored the possible regulatory mechanism of the DHRS4 gene cluster in mammalian evolution by analyzing the promoter sequence, methylation of CpG islands, and RNA expression of the DHRS4 gene cluster in mice, chimpanzees, and humans by bioinformatics prediction, bisulfite sequencing PCR, and real-time reverse transcriptase-PCR. The results indicated that the DHRS4 gene was actively expressed in the three model species. The RNA level of DHRS4L1 was much lower than those of DHRS4 and DHRS4L2, and expressed lower homologous sequence identity to DHRS4 and DHRS4L2. DHRS4L2, the latest evolutionary copy of the DHRS4 gene in mammals, received a high promoter prediction score, and was the only copy of the DHRS4 gene cluster presenting hypermethylated CpG islands in the promoter region. An analysis of the relationship between the promoter characteristics and RNA expression of the DHRS4 gene cluster indicated that the development of CpG islands, in addition to the promoter sequence, during mammalian evolution could modulate the dose compensatory regulation of the copy number-varied DHRS4 gene cluster.