Anuran skin contains a calcified dermal layer, referred to as the Eberth-Kastschenko (EK) layer, which is found between the stratum spongiosum and the stratum compactum. Although it is established that some anuran species possess the EK layer, little is known about this layer from the standpoint of evolutionary and developmental biology. We conducted a morphological analysis by staining the dorsal skin from many species with alizarin red S to investigate the calcified layer. This layer was observed in all of the anurans tested, as well as in fishes and one species of caecilian with dermal scales, but not in urodeles, amniotes, or a scaleless caecilian. All of the investigated species with dermal scales exhibited a calcified layer in their dermis, while the anurans showed the EK layer, but no scales. We also analyzed the expression of genes related to scale formation (sparc, mmp9, and mmp2) in the dorsal skin of X. tropicalis. These genes were highly expressed at the metamorphic climax stage, which preceded the deposition of calcium. Furthermore, we examined the gene expression profile of amelogenin, the major protein found in the enamel matrix of the developing tooth. In X. tropicalis, amelogenin was upregulated in the skin at the climax stage and was expressed in the adult dermis at a high level. These data provide the first experimental evidence of the expression of amelogenin in the skin. These findings will lead to a better understanding of the developmental formation of the EK layer and the function of amelogenin.