Opuntia genus belongs to the Cactaceae and includes a range of species from 75 to 250 originated from the Americas. Several Opuntia species represent useful sources of forage in many arid areas, and their cultivation is drastically increasing. However the taxonomic classification of these species is complicated by the lack of reliable morphological descriptors, the relevant phenotypic plasticity, and the frequency of natural hybridization and of polyploidy. In this study 31 genotypes selected for forage production including Opuntia ficus-indica and some related species were analyzed using SSR markers to establish their genetic variability and to elucidate phylogenetic relationships among cultivated genotypes and related species. The analysis of six loci allowed to univocally identify most of the accessions and confirmed the fact that their taxonomical classification is not congruent with the observed patterns of genetic diversity. Most of the accessions selected for forage production were grouped exhibiting a narrow genetic variation level and clustered separately from two reference O. ficus-indica genotypes, used for fruit production in Mediterranean area. NeighbourNet analysis displayed a low level of diversification among the forage Opuntia from Brazil suggesting they probably have common ancestors. This information will be useful to plan future breeding strategies aimed at the selection of improved genotypes to be cultivated in different areas.