Hylodinae leptodactylids(sensu Lynch 1971) form a group of diurnal frogs, which is hypothesized on the basis of morphological traits to be the closest relatives of the dendrobatid frogs. Our study describes ultrastructural characteristics of sperm from three hylodine species(Hylodes phyllodes, Crossodactylus sp. n. andMegaelosia massarti) to reassess the intergeneric relationships within the Hylodinae, as well as the supposed relationship between the Hylodinae and Dendrobatidae. The ultrastructure of the sperm is very similar among the three species and is indicative of its conserved nature within the Hylodinae. The structure of the acrosomal complex was very similar to that of other leptodactylid species, to most of the remaining species included in the Bufonoidea lineage, and also to that observed in the dendrobatid species examined so far. Since such a structure has been considered a plesiomorphic trait, it contributes little to our understanding of the relationships between the Hylodinae and Dendrobatidae. The flagellar apparatus ofCrossodactylus sp. n. is very similar to that of most leptodactylids. The sperm ofMegaelosia massarti andHylodes phyllodes display a distinctive condition in their axial and juxtaxonemal fibers. This distinctive flagellar condition expands the already known variability in sperm structure within the Leptodactylidae.