The family Phyllostomidae belongs to the most abundant and diverse group of bats in the Neotropics with more morphological traits variation at the family level than any other group within mammals. In this work, we present data of chromosome banding (G, C and Ag-NOR) and Fluorescence In Situ Hybridization (FISH) for representatives of Rhinophylla pumilio Peters, 1865 collected in four states of Brazil (Amazonas, Bahia, Mato Grosso and Pará). Two karyomorphs were found in this species: 2n=34, FN=64 in populations from western Pará and Mato Grosso states and 2n=34, FN=62 from Amazonas, Bahia, and northeastern Pará and Marajó Island (northern). Difference in the Fundamental Number is determined by variation in the size of the Nucleolar Organizer Region (NOR) accompanied with heterochromatin on chromosomes of pair 16 or, alternatively, a pericentric inversion. The C-banding technique detected constitutive heterochromatin in the centromeric regions of all chromosomes and on the distal part of the long arm of pair 15 of specimens from all localities. FISH with a DNA telomeric probe did not show any interstitial sequence, and an 18S rDNA probe and silver staining revealed the presence of NOR in the long arm of the pair 15, associated with heterochromatin, and in the short arm of the pair 16 for all specimens. The intra-specific analysis using chromosome banding did not show any significant difference between the samples. The comparative analyses using G-banding have shown that nearly all chromosomes of R. pumilio were conserved in the chromosome complements of Glossophaga soricina Pallas, 1766, Phyllostomus hastatus Pallas, 1767, Phyllostomus discolor Wagner, 1843 and Mimon crenulatum Geoffroy, 1801, with a single chromosomal pair unique to R. pumilio (pair 15). However, two chromosomes of M. crenulatum are polymorphic for two independent pericentric inversions. The karyotype with 2n=34, NF=62 is probably the ancestral one for the other karyotypes described for R. pumilio.
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