Three species of the genus Megaelosia (Megaelosia massarti, Megaelosia boticariana, and Megaelosia lutzae) were studied karyotypically using conventional Giemsa staining, C-banding, and NOR techniques. Megaelosia lutzae had 2n = 32 chromosomes, a new diploid number for this genus and for the subfamily Hylodinae. Two morphotypes of B-chromosomes were found in two specimens of M. massarti. C-banding patterns and NOR location varied among the species. Nevertheless, some chromosome pairs, as well as the NOR-bearing secondary constriction and the large pericentromeric C-block on the short arm of chromosome 10, were consistent among the three species. Megaelosia differs markedly from other Hylodinae and Dendrobatidae in terms of karyotype.