Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a peptide growth factor and member of the large TGF-β family of growth and differentiation factors. Using RT-PCR and RACE, this paper isolated two AMH cDNA from Oreochromis aureus which was different in 3′UTR. One was 621 bp, the other was 168 bp. They differed by only 2 bp excluding the different sequence length. The same 5′ UTR was 27 bp and the same ORF was 1 545 bp, which encoded 514 amino acids containing the two domains characteristic of the AMH protein family: the TGF-β domain 93 amino acids and the AMH-N domain 237 amino acids. Homology analysis showed that AMH shared little conservation. O. aureus was more similar to the orthologous regions in other fishes (32%-60%) than to the corresponding regions of mammalian AMH genes (19%-21%). However, the TGF-β domain shared relatively high conservation. The similarity was 54%-72% between O. aureus and other fishes. Comparing the sequence of cDNA to its genome revealed AMH gene consisted of six introns, which was similar in zebrafish and different in mouse and human due to the absence of two additional introns (intron Ⅰand intron Ⅵ). Analysis of O. aureus AMH promoter showed the presence of several putative transcription factor binding sites: SRY, SF-1, GATA-4, Sox5, Sox9. Real time RT-PCR (Taqman probe) was used to detect the AMH expression in adult fish different tissues including testis, ovary, brain, liver, muscle and heart, and the AMH expression level in different development stages including larva, juvenile, adult fish. The results showed that AMH was only expressed in ovary and testis. Comparing the AMH expression level in the three development stages, we concluded that AMH was not expressed in larva, and the amount of gene transcripts was significantly higher in male than in female in juvenile and adult fish, and it was 10 times in male juvenile as in male adult fish.