Chromosome numbers are determined for 524 individuals belonging to 107 taxa, including nothotaxa, within Cactaceae. Chromosome morphology was uniform within and among species. Nine unnamed nothotaxa and one previously named nothotaxon, Cylindropuntia ×vivipara, have chromosome numbers reported for the first time, and new chromosome numbers are reported for three taxa. Five putative Cylindropuntia hybrids are reported here for the first time: C. alcahes var. gigantensis × C. lindsayi, C. bernardina × C. echinocarpa, C. leptocaulis × C. ramosissima, C. echinocarpa × C. ramosissima, and C. echinocarpa × C. ganderi var. ganderi. Two putative Opuntia hybrids of less-certain origin are reported here: O. azurea var. discolor × O. rufida, and O. chlorotica × O. engelmannii. Two putative hybrids are reported for Echinocereus: E. arizonicus subsp. arizonicus × E. bonkerae subsp. bonkerae and E. coccineus subsp. coccineus × E. yavapaiensis. All chromosome number determinations reported to date are tabulated for Cylindropuntia, Grusonia, and selected taxa within Echinocereus. The significance of these data is discussed, including apparent correlations among polyploidy, subdioecy, sexual reproduction, and apomixis. We introduce six new combinations, one nothospecies designation, and a lectotypification for one taxon.