Complementary DNAs encoding precursor molecules of the β subunits of three pituitary glycoprotein hormones (LH, FSH, and TSH) of the Japanese toad ( Bufo japonicus) were isolated and sequenced. Unexpectedly large numbers of single nucleotide substitutions were found in all three β subunit cDNAs. The eight isolated LH β precursor cDNA clones were classified into six forms of nucleotide sequence, with four nucleotide substitutions each in the apoprotein coding region and in the 3 ′ untranslated region (UTR). In the deduced amino acid sequence, the LH β subunit showed two forms with a single amino acid substitution. The seven isolated FSH β subunit cDNAs were classified into two forms, which differed from each other at 11 positions in the 3 ′ UTR. The six isolated TSH β subunit clones were classified into four forms with 2 and 5 nucleotide substitutions in the signal peptide and apoprotein coding regions, respectively. However, all the substitutions in the apoprotein coding region were silent. The substitution in the signal peptide coding region could produce three forms of signal peptide. Amino acid sequence comparison revealed that the toad LH β subunit is more similar to the fish GTH II β subunit than to mammalian and avian LH β subunits. We found that the toad LH β subunit molecule is a partial chimera of LH and FSH; amino acid residues located in 36th to 42nd and 96th to 99th are identical or similar to those of not LH- but FSH-β subunit in mammalian, whereas it is more similar to LH- than FSH-β subunit in total. We also found that the toad FSH β subunit is more similar to the fish GTH II β subunit than to the fish GTH I β subunit and that the toad TSH β subunit is more similar to tetrapod TSH β subunits than to fish TSH β subunits.