Species of Protopolystoma are monogenean flukes that only infect allopolyploid hosts in the anuran genus Xenopus. Multivariate analyses of morphometric sclerite characters in the nominal species Protopolystoma simplicis suggest that morphologically distinguishable populations occur in the tetraploid host, Xenopus laevis victorianus, and in each of the octoploid hosts, X. vestitus and X. wittei. The species-level divergence of a lineage specific to X. laevis is supported by sequence variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Protopolystoma simplicis from X. laevis is redesignated P. microsclera n. sp., with P. simplicis being retained for populations in octoploid hosts. This division is consistent with large differences in egg hatching schedule, fixed differences at the mannose-6-phosphate isomerase and fumarate hydratase loci, and host-specificity in experimental analyses. Although the respective P. simplicis populations in X. vestitus and X. wittei also show significant diversity in allozyme expression, morphometrics and egg hatching schedule, they are retained in the same species because their level of mitochondrial DNA divergence is similar to that found within other Protopolystoma species. The consequences of splitting P. simplicis for a recent interpretation of the origin of Protopolystoma faunas in octoploid Xenopus spp. is discussed.