There is genetic variability among prickly pear species, which makes it possible to use divergent accessions in genetic improvement programs. From this perspective, this study aimed to characterize and recommend Opuntia spp. accessions as potential progenitors for genetic breeding programs conducted by the consortium of institutions associated with the National Institute of the Semi-Arid Region (INSA), Brazil. The experiment was carried out in the Laboratory of Biotechnology and Plant Breeding at the Center of Agricultural Sciences, Federal University of Para & iacute;ba, State of Para & iacute;ba, Brazil. Seventy prickly pear accessions from the Active Germplasm Bank of the National Institute of the Semi-Arid Region were used and characterized using inter simple sequence repeat (ISSR) markers with 13 primers. For each primer, the number of amplified bands, number of loci, polymorphic information content, marker index, resolving power, expected heterozygosity, and inter- and intraspecific diversity were estimated through non-parametric variance analysis. The unweighted pair group method with arithmetic mean (UPGMA) method was used to cluster the accessions. The majority of genetic diversity is intraspecific (82.08%). Accessions 45, 50, 60, 68, and 81 (O. cochenillifera), 20, 47, and 79 (O. ficus-indica), and 53 (O. stricta) are recommended as potential progenitors for the genetic improvement program of this crop due to their genetic diversity.