Xoconostle or acidic prickly pear is an important fruit in Mexico; it is produced by a group of Opuntia plants known for their nutritional qualities and adapted to harsh environmental conditions. In this study, we report for the first time the estimation of genetic diversity within a set of 24 xoconostle accessions using inter simple sequence repeats (ISSR) markers. Five ISSR primers were selected to detect DNA polymorphisms in these plants. Broad genetic diversity was detected (0.41-0.82, Dice coefficient) and the unweighted pair group method with the arithmetic averaging (UPGMA) analysis allowed discriminating all genotypes and enabled their clustering into six groups; the 'Matizado' accession was significantly divergent from all tested genotypes. In addition, as shown by the clustering analysis and principal component analysis (PCA), the tested genotypes did not group according to their origin, to culture conditions, or to the current species classification. This work, on one hand, demonstrates the high genetic variation among genotypes of xoconostles, and on the other hand, it suggests a taxonomic revision and raises doubts about the number of species reported for these plants.