We studied the cariotype of Holochilus venezuelae from 28 individuals (14 males and 14 females). Chromosome preparations were obtained by applying colchicine to live animals, followed by hypotonic shock, acetic methanol fixation and Giemsa staining of bone marrow cells. Metaphase configurations were photographed with a M-20 Wild automatic photomicroscope, and 10 karyotype from each animal were prepared from enlarged prints. Idiograms were calculated from the mean length of each chromosome pair and expressed as percentage of the length of the haploid autosome set plus one X chromosome. Results indicate the presence of six karyomorphs and the most frequent karyotype, considered basic for the species, was 2n = 44 (NF = 56), which consists of seven pairs of metacentric chromosomes (five large, one small and one microchromosome), comprising 59.5% of the haplid set; fourteen acrocentric pairs (nine small and five microchromosomes) representing 32.0% of the set; and the sexual pair, which is acrocentric, represent 6% (X) and 2.5% (Y) of the haploid set. The chromosome polymorphisms can be interpreted as the result of Robertsonian changes (fusion) and from the presence of accessory chromosomes (supernumerary). The karyotypical characteristics of the population sample studied suggest the existence of reproductive isolation through chromosomal hybrid sterility with H. brasiliensis (2n = 56 and 2n = 50), which means that H. venezuelae can be considered an allospecie.