Total RNA was extracted from the pituitary gland of half-smooth tongue sole Cynoglossus semilaevis Günther, and the follicle stimulating hormone (FSH) was cloned by using RT-PCR and RACE technology for the first time. The cDNA of FSH is 541bp, and the open reading frame was 393bp encoding peptides of 130 amino acids. This cDNA sequence was submitted to GenBank with Accession No. JQ277933. One N-glycosylation site was found in the deduced amino acid sequence. Multiple amino acid sequences alignments were performed among vertebrates including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. It showed that half-smooth tongue sole FSH mature peptide was more similar to their orthologs of Pleuronectiformes and Perciformes which was 42%-49% identity than to Cypriniformes and mammalian which was 27%-31% identity. The pairwise distances were computed and phylogenetic trees were constructed using NJ method based on the codon sequences of the chosen species by MEGA 4.0 software. The result indicated that the FSH of half-smooth tongue sole was clustered together with other fish FSH, and the half-smooth tongue sole gene shared high homology with teleost. Real-time PCR analysis showed that FSH mRNA was expressed at the highest level in pituitary. Also strong amplification signals were detected in all other tissues, especially brain and gonad. FSH mRNA of half-smooth tongue sole expressed widely in non-pituitary tissues may indicate that it has more physiological functions.