We performed karyotype analyses on Hylocereineae species. Epiphyllum phyllanthus, Selenicereus aff. megalanthus and S. aff. setaceus were diploid, with 2n = 22 chromosomes, whereas S. setaceus was tetraploid. Karyotypes were symmetrical with the formulas 11 m in E. phyllanthus, S. aff. setaceus and S. aff. megalanthus, and 22 m in S. setaceus. Chromosomes were small with the average length being 2.76 μm. Diploid species had two terminal satellites and tetraploids had four terminal satellites. The only bands associated with NOR were found in all specimens and were CMA /DAPI^- bands. Pericentromeric heterochromatin was found in S. aff. megalanthus. The locations of the 18-5.8-26S rDNA sites in all species coincided with CMA bands. The signals of the 5S rDNA gene were located in paracentromeric regions on at least one locus in all taxa. Additional 5S loci were found in all species, with the exception of E. phyllanthus. One pair of 5S sites was co-localized with 18-5.8-26S loci in S. aff. setaceus and tetraploid S. setaceus, which is a new position for Cactaceae. The 18-5.8-26S rDNA loci seem to be highly conserved, in contrast with the 5S rDNA loci that were scattered throughout the genome.