Two types of cDNA (GTH-Iβ and -IIβ) encoding the β subunit of masu salmon (Oncorhynchus masou) gonadotrophin were cloned using the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequence of GTH-Iβ cDNA was 469 bp long, encoding 137 amino acids, and GTH-IIβ cDNA was 476 bp long, encoding 142 amino acids. These two masu salmon β 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-Iβ and GTH-IIβ respectively. These time values are markedly inconsistent with the evolutionary time (30 million years) estimated from fossil records and an isozyme study. Southern blot analyses showed that the Iβ gene restriction fragment lengths differed among five teleosts, whereas, with one exception, the IIβ gene showed well conserved patterns. Therefore, the GTH-Iβ gene may have diverged at a faster rate than the GTH-IIβ gene.