Two types of cDNA (GTH-I beta and -II beta) encoding the beta subunit of masu salmon (Oncorhynchus masou) gonadotrophin were cloned using the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequence of GTH-I beta cDNA was 469 bp long, encoding 137 amino acids, and GTH-II beta cDNA was 476 bp long, encoding 142 amino acids. These two masu salmon beta subunit types showed low homologies of 52% (nucleotide sequence) and 33% (amino acid sequence). The evolutionary interval between masu and chum salmon was estimated to be 5.65 and 1.43 million years by comparing GTH-I beta and GTH-II beta respectively. These time values are markedly inconsistent with the evolutionary time (3.0 million years) estimated from fossil records and an isozyme study. Southern blot analyses showed that the I beta gene restriction fragment lengths differed among five teleosts, whereas, with one exception, the II beta gene showed well conserved patterns. Therefore, the GTH-I beta gene may have diverged at a faster rate than the GTH-II beta gene.