Two subtype estrogen receptor β (ERβ1/β2) cDNAs were obtained from blue tilapia, Oreochromis aureus by Reverse Transcription Polymerase Chain Reaction (RTPCR) and RACE. The full length cDNA sequence of ERβ1 was 4 262 bp in length, containing a 5′ Untranslated Regions (UTR) of 239 bp, 3′ UTR of 2 349 bp and an open reading fame (ORF) of 1 674 bp which encoded a putative protein of 557 amino acids with an estimated molecular weight of 62 ku. The ERβ2 cDNA was 2 506 bp in length with 393 bp of 5′ UTR, 109 bp of 3′ UTR and an ORF of 2 004 bp which encoded a putative protein of 668 amino acids with an estimated molecular weight of 74 ku. The deduced amino acid sequence of ERβ1 of blue tilpia possessed 99.1% similarity with the counterpart of nile tilapia, O. niloticus, and similarities with other Perciformes fishes were from 82.6% to 94.2%. The ERβ2 amino acid sequences of blue tilpia shared 98.7%, 81.8%, 76.3%, 64.7% and 55.0% identities with nile tilapia, Micropterus salmoides, Oncorhynchus mykiss, Fundulus heteroclitus and Danio rerio respectively. Blue tilpia ERβ1/β2 were clustered with nile tilapia ERβ1/β2 respectively in the phylogenetic tree. ERβ1/β2 mRNA expressions between male and female were compared by RealTime PCR (RTPCR). ERβs were expressed ubiquitously and differently in ten tested tissues. ERβ1 was significantly expressed in ovary, testis, liver, kidney and intestinal tissues compared with other tissues (P<0.05). ERβ2 was highly expressed in testis, liver and kidney (P<0.05). In ovary, testis, intestine and hypothalamus, expression of ERβ1 was significantly higher than that of ERβ2, and it was 164 times of ERβ2 in ovaries. After 17 betaestradiol (E₂) was injected for 24 h, ERα/β1 mRNA in hypothalamus of male blue tilpia was increased, of which ERα increased significantly. Expression of ERα in hypothalamus increased significantly in 7.5 mg/kg E₂ treated group, while no significant change was observed for ERβ2. In pituitary, expression of ERα/β1/β2 in the low and middle E₂ concentrations (2.5, 5.0 mg/kg) was downregulated, but upregulated in high E₂ concentration group (7.5 mg/kg), and significant difference was found for ERα(P<0.05) . ERβ2 expression was increased in testis significantly (P<0.05) in middle and high E₂ concentration groups (5.0, 7.5 mg/kg). No significant change was observed for ERα/β1 expression. The different distribution of the ERβ1/β2 mRNAs and different regulation of ERα/β1/β2 mRNA in hypothalamus, pituitary and testis by E₂ indicated that various biological functions existed among these three ER subtypes in blue tilpia.