The family Hylodidae is composed of 46 species distributed in three genera: Crossodactylus (13 species), Hylodes (26 species), and Megaelosia (seven species). Although the monophyly of the Hylodidae is supported by previous molecular phylogenetic inferences, the monophyly of Megaelosia and/or Hylodes has been questioned. Crossodactylus and Hylodes share the plesiomorphic diploid chromosomal number 2n = 26, whereas in the species of Megaelosia as karyotyped up to now, diploid numbers have ranged from 28 to 32. Here, we expand to six (of seven) the number of species of Megaelosia sampled in a phylogenetic approach based on mitochondrial and nuclear DNA sequences and describe the 26-chromosome karyotype of M. goeldii for the first time. Our results provide an improved perspective on the relationships among the frogs described previously in the genus Megaelosia, and we propose a new taxonomic arrangement, in which Megaelosia is a monotypic genus, with a new genus being described to accommodate the remaining species allocated previously to Megaelosia. We also confirm that 2n = 26 is the plesiomorphic diploid number in the Hylodidae, and conclude that an increased diploid chromosome number, together with two morphological traits and one ethological characteristic, are potential synapomorphies of the new genus.