Abstract Ameloblastin (AMBN) is an enamel sheath protein that presumably has a role in determining the prismatic structure of growing enamel crystals. To investigate the relationship between the molecular evolution of the AMBN gene and development of enamel prismatic structures, it is considered to be of great significance in the identification of homologues of the AMBN genes in nonmammals whose teeth lack an enamel prismatic structure. Several clones containing AMBN cDNA were isolated from an African clawed toad tooth cDNA library by screening with a polymerase chain reaction (PCR) method. Sequence analysis of the clones revealed that they were derived from different genes ( toad-A and toad-B), which were found to contain ORFs encoding 408- and 352-amino-acid proteins, respectively. The N-terminal part of the toad AMBN proteins and the phosphorylation motif for casein kinase II, as well as several features, were found to be highly conserved throughout the evolution of tetrapods. Exon–intron boundaries were shared by toad and caiman genes with the exception of exons 6, 7 and 10 while human and caiman genes shared them exclusive of exons 8 and 9 which have been found only in the human. As for exon 7, it was absent in both toad genes. Moreover, the AMBN genes were transcribed only in the upper jaw, presumably in teeth. These results may provide useful information for investigation of the evolution of enamel.