Representatives of the Cactaceae subfamilies Pereskioideae and Opuntioideae from northeastern Brazil were studied using banding with the fluorochromes, CMA3 and DAPI, as well as with fluorescent in situ hybridization using 45S and 5S rDNA probes to identify the distributions of their heterochromatin and rDNA sites. Pereskia aculeata, P. bahiensis, P. grandifolia (Pereskioideae), Brasilopuntia brasiliensis, Tacinga funalis, and T. palmadora showed 2n = 22, while Opuntia dillenii showed 2n = 44, and O. ficus-indica 2n = 88. The karyotypes of all of the species were symmetric, with average chromosome lengths varying from 1.94 lm in O. dillenii to 3.17 lm in P. aculeata. One pair of terminal CMA? bands corresponding to NORs occurred in all of the diploid cytotypes (except O. ficus-indica, which has two pairs of terminal CMA? bands) as well as in O. dillenii (tetraploid). CMA? bands were also observed in the interstitial region of the long arm of a chromosome pair in B. brasiliensis, while a number of variable proximal bands were observed on three chromosome pairs in O. dillenii and on most of the chromosomes of O. ficus-indica. The 45S rDNA sites corresponded to the terminal CMA? bands, while the 5S rDNA sites were located in the interstitial regions of the long arms of the chromosome pairs of P. aculeata, P. bahiensis, P. grandifolia, and B. brasiliensis. Our data, and earlier publications, suggest that the subfamily Opuntioideae can be characterized as having proximal/interstitial CMA? heterochromatin in at least one chromosome pair (except in Tacinga). The absence of proximal heterochromatic bands, however, appears to be a synapomorphy of the basal lineages of Cactaceae (subfamily Pereskioideae ? Maihuenioideae), suggesting that karyotypes with heterochromatin restricted to the terminal region of a chromosome pair (45S rDNA) represent a plesiomorphic character of the family.