Abstract: Maturation inducing hormone (MIH) plays pivotal roles in gamete maturation, ovulation and spermiation of fish. In most teleost species, 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is the major MIH. The steroidogenic enzyme, named 20β-hydroxysteroid dehydrogenase (20β-HSD), is responsible for DHP production in several teleost species. To date, only one copy of 20β-hsd has been characterized in vertebrates, including fish. In this study, two copies of 20β-hsd genes were identified in genomes of several fish species. Phylogenetic and syntenic analyses revealed that the duplication of 20β-hsd gene may not result from the teleost specific genome duplication (TSGD). Multiple-alignment re- sults indicated that these two enzymes might possess similar spatial structures and co-enzyme/substrate binding motifs, whereas, their catalytic activities might differ. The duplicated copy of 20β-hsd was further cloned from yellow catfish (Pelteobagrus fulvidraco). Tissue distribution results showed that, 20β-hsd Ⅰ mRNA was mainly expressed in testis, ovary, head kidney and kidney, while 20β-hsd Ⅱ mRNA was mainly located in testis, ovary, heart, head kidney, kidney, intestine, pituitary and liver. Seasonal expression results revealed that, lower 20β-hsd Ⅰ expression and higher 20β-hsd Ⅱ expression were observed in male fish during reproductive time window (in May). Three hours after injection of hCG (1000 IU/kg body weight), the expression of 20β-hsd Ⅰ was up-regulated and then suppressed signifi- cantly, while the expression of 20β-hsd Ⅱ was up-regulated in a time dependent manner in male fish. These results demonstrated the differential regulation pattern of 20β-hsd Ⅰ and 20β-hsd Ⅱ in response to hCG.