Characterizing the diversity and lability of the amphibian sex chromosomes holds key to understand what drives sex chromosome turnovers and assess the role of sex-linked genes in reproductive isolation and speciation. Here, we show that the heterogametic transition previously reported between the hybridizing toads Bufo bufo (ZW) and Bufo spinosus (XY) is nonhomologous, potentially implicates key genes of the vertebrate sex determination cascade (SOX9, DMRT1, and AMH), and is characterized by a much shorter ZW than XY segment. Integrating this information with published hybrid zone data suggests that both sex chromosomes resist interspecific introgression more than autosomes. These observations substantiate that sex chromosome turnovers preferentially involve chromosomes that host conserved sex-determining genes, imply heterochiasmy as a key factor of sex chromosome differentiation, and are consistent with a large sex chromosome effect, an empirical rule of speciation that is not expected with homomorphic sex chromosomes.