Background: The DHRS4 gene encodes an NADP(H)-dependent retinol dehydrogenase /reductase.DHRS4 and two genes (DHRS4L2 and DHRS4L1) that recently arose from gene duplication in human, an antisense transcript AS 1DHRS4, devoted of the DHRS4 gene negatively regulates expression of all three genes.In order to analysis the evolution of the DHRS4 gene cluster and antisense AS1DHRS4, bioinformatics methods were performed to analysis it.Methods: Infernal-l.0.2 was run to searching DNA sequence databases for RNA structure and sequence similarities homologous sequences of AS1DHRS4 in fifteen species.Multiple sequence alignments and evolution analysis were completed by ClustalX2.0.Mega5.0 was used to estimate rates of molecular evolution.3-RACE and 5-RACE were conducted to obtain the full length of CO048964.We also transfected rhesus monkey siRNA to knockdown CO048964, and detected the knockdown effect by real-time PCR.Results: Sequence alignment of Infernal-l.0.2 shows that DHRS4 sequences are conserved in vertebrates.From the sequence alignments, we realized that most primates have a single duplication.However, human and rhesus monkeys contain two duplications, while marmosets and other vertebrates contain only one copy.Sequence alignment showed that the AS1DHRS4 does not exist in mouse and rat even though both genomes have high similarity with the human genome.AS1DHRS4 is conserved in all primates except in marmosets, but exists in the microbat.We found an EST sequence CO048964 in rhesus monkey that transcribed at the opposite chain of the DHRS4 gene cluster.Its exon structure is extremely different from AS1DHRS4.The rhesus monkeys full length CO048964 was obtained through 3-RACE and 5-RACE in Frhk-4 cell line RNA, and devoted it rheAS 1DHRS4.GenBank ID: JQ686618, JQ686619, JQ686620.Knockdown of rheAS 1DHRS4 in Frhk-4 cell led significant down regulation of DHRS4, which is opposite to AS 1DHRS4 in human cells.Conclusions: The sequence of DHRS4 gene is conserved among the fifteen species, but the DHRS4 gene cluster is not evolutional conserved, higher and more complicated animals seem to have more copies of DHRS4.The AS 1DHRS4 antisense transcript emerges in primates but evolved to have different regulatory on DHRS4 gene, suggesting that various regulatory mechanisms can evolve from long nocoding RNAs .