The genetic diversity for twenty species belonging to four genera of Cacataceae (Rebutia, Aylostera, Mediolobivia and Sulcorebutia) was analyzed employing taxonomic methods, chromosomes number and RAPD markers. The botanical classification that describes the phenotypic aspects of different characters, such as plant diameter, number of spines/areole, mean spines length, flower diameter, and flower color, was employed. Chromosome analysis revealed both diploids and polyploids in the studied species. Polyploidy was represented by two tetraploids species (S. crispata and R. kupperiana var. spiniflorum, 2n =4x =44) and by five triploids species (R. pseudodeminuta var. schumaniana, R. senilis var. liliacinorosea, R. calliantha, R. donaldiana and M. diersiana, 2n =3x =33). Thirteen species were diploid with 2n =2x =22. The total number of amplified bands was 448, and 400 bootstrap samples for generating the dendrogram, were used. The dendrogram indicates the diversity of the genotypes, which are grouped into four distinctive large groups. Almost all groups include species from the Rebutia, Aylostera, Mediolobivia and Sulcorebutia genera, which clearly share a common ancestor. M. diersiana and R. donaldiana, two triploid species (2n=3x=33) formed the same subgroup in the frame of the dendrogram. In addition, those species also present common phenotypical peculiarities like: colour of flower (orange), diameter of flower (2.5 cm). The taxonomic methods were less precisely, regarding to assess genetic variation, comparing with cytological methods, and molecular markers methods.