In mammals, leptin is an endocrine hormone involved in the regulation of feeding and energy metabolism, and glucocorticoids were previously reported to induce leptin gene expression. However, the link between glucocorticoids and leptin in fish has not been investigated. Goldfish are tetraploid teleosts because of genomic replication events (FSGD or 3-R), and some specific genes have quadrupled. In this study, two goldfish leptin-B genes, namely, leptin-BI (GenBank: ON468243) and leptin-BII (GenBank: ON468244), were cloned. The cDNA sequences of leptin-AI (GenBank: FJ534535.1) and leptin-AII (GenBank: FJ854572.1) have been reported by our team previously. Goldfish leptin-AI, -AII, -BI and -BII genes are located on chromosomes 43, 18, 29 and 4, respectively. In goldfish, leptin-B proteins shared approximately 30 % amino acid identities with leptin-A proteins and only 20 % with human leptin; however, either goldfish leptin-A or leptin-B showed high amino acid homology between themselves, 79.29 % and 82.74 %, respectively. The four leptin genes in goldfish are all highly conserved in three-dimensional (3-D) protein structures and gene structures. A tissue expression study showed that both the A and B forms of goldfish leptins are predominantly in the brain and liver. The effects of glucocorticoids on four leptin transcripts expression in goldfish were investigated by in vivo intraperitoneal injection and in vitro cell incubation approaches. The results showed that cortisol inhibited hepatic leptin-A transcript expression in a dose-dependent manner, while cortisol stimulated hepatic leptin-B mRNA expression, and RU486 (glucocorticoid receptor inhibitor) abolished cortisol’s effect on hepatic leptin-A and leptin-B mRNA expression. These results reflected the differential regulation of leptin-A and leptin-B by glucocorticoids in goldfish, which implied that leptin-A and leptin-B may mediate different functions in goldfish.