We conducted a chromosome banding analysis (heterochromatin, nucleolus organizer regions, DAPI fluorescence, distamycin A/mithramycin fluorescence) of six phylogenetically basal dendrobatid frog species (Colostethus chalcopis, C. leopardalis, Mannophryne herminae, M. neblina, M. olmonae, M. trinitatis). With the exception of C. chalcopis (2n = 22), all examined species had a chromosome complement of 2n = 24 chromosomes. The C- and Q-band analyses showed that constitutive heterochromatin is present at the centromeres of all species, with Q⁻-regions occurring at the positions of the nucleolus organizer regions (NORs) of C. leopardalis, M. olmonae, and M. trinitatis. The C-band polymorphisms were detected in M. herminae and M. neblina on chromosome No. 6 and in M. herminae on chromosome No. 7. Silver-staining and distamycin A/mithramycin fluorescence resolved a single pair of NORs in each species. The DAPI fluorescence revealed pericentromeric bands on chromosome No. 1 and 5 in M. trinitatis and on chromosome No. 1 and 4 in C. chalcopis. Chromosome data clearly allow a distinction between M. olmonae and northern Trinidadian M. trinitatis, laying to rest arguments that M. olmonae may not be a good species. In conjunction with chromosome information found in the literature, our data confirm that chromosome complements of phylogenetically basal dendrobatids can be quite variable, with both 2n = 24 and 2n = 22 complements present. Discrepancies in the karyotypes of M. trinitatis from near Caracas, Venezuela, and from the Northern Range of Trinidad indicate the possibility of cryptic species within M. trinitatis. The notion that a reduction in chromosome number by up to three chromosome pairs has taken place among Dendrobatidae from a putative ancestral 2n = 24 karyotype is shown to be congruent with current molecular hypotheses of dendrobatid relationships.