) genes from spotted halibut ()by isolating their cDNAs. This species expressed three molecular forms of GnRH in the brain: chicken type GnRH-), seabream type GnRH (sbGnRH), and salmon type GnRH (sGnRH). Each cDNA encoded a signal peptide(SP), GnRH, and a GnRH-associated peptide(GAP), which was connected to GnRH by a Gly–Lys–Arg sequence. consisted of 85 amino acid residues, and the cDNA was 568 bp in length, consisting of a 141 bp 5′UTR, a 255 bp open reading frame (ORF), and a 169 bp 3′UTR. The sGnRH consisted of 90 amino acid residues and was 457 bp in length. It consisted of a 41 bp 5′UTR, a 270 bp ORF, and a 143 bp 3′UTR. The sbGnRH precursor consisted of 98 amino acid residues, and the cDNA was 381 bp in length and consisted of a 48 bp 5′UTR, a 294 bp ORF, and a 36 bp 3′UTR. Homology analysis of the deduced acid residues of the GnRH precursors revealed that the three spotted halibut genes had the highest identity with homologs from pleuronectiformes, followed by perciformes, salmoniformes, and anguilliformes. Phylogenetic analysis indicated that the multiple molecular forms of spotted halibut GnRHs shared the closest evolutionary relationship with counterparts from pleuronectiformes. The tissue and sex specific expression of these three genes was determined using real time PCR. Expression was highest was detected exclusively in the brain whereas sbGnRH was expressed globally in both sexes. sGnRH was detected in the pituitary and gonad (in addition to the brain tissue) in females, but not in the gonad of males. The expression pattern of all three genes was sex-specific with females having higher mRNA levels than males. We evaluated the changes in mRNA expression in the female brain during ovary maturation. mRNA levels increased significantly (<0.05) after August (vitellogenic stage), peaked in December (prespawning stage) 2009, and then decreased by April 2010 (postspawning stage). Conversely, there was no change in either Our results confirmed that spotted halibut expressed three GnRH isoforms and sbGnRH may be the most important form. Our observations provide insight into the endocrine control of reproduction in the spotted halibut.