Comparison of advertisement calls of Eleutherodactylus guentheri from different localities in southern and southeastern Brazil revealed considerable acoustic variation among populations. Because of significant differences in call parameters, we conclude that the southern form is a distinct species, for which the name Hylodes henselii Peters, 1870, is available. Examination of the holotypes of H. henselii and Hylodes guentheri Steindachner, 1864, along with recently collected material, showed that both species are morphologically similar, possibly sibling species. Eleutherodactylus henselii is the southernmost member of the genus Eleutherodactylus occurring in subtropical Atlantic rain forest and Araucaria forest of the Brazilian states of Rio Grande do Sul and Santa Catarina, and Misiones, Argentina. Bioacoustic data suggest that at least one additional cryptic species is involved in the Eleutherodactylus guentheri group.