In the present work, routine alkaloid screening revealed the presence of alkaloids in several species of the genus Opuntia. Alkaloids were crystallized as their hydrochloride salts from the screening extract of O. stanlyi var. kunzei and from a large-scale extraction and PLC of O. schottii. Non-crystalline alkaloids were identified by TLC (cochromatography in five solvents on SGG). Known β-phenethylamines were identified in eleven species of the subgenus Cylindropuntia (cholla). Alkaloids, including primary and secondary amines of undetermined chemical class, were detected in five of the six investigated species of the subgenus Opuntia (prickly pear); none could be identified by TLC comparison with a series of reference cactus alkaloids, and this subgenus clearly lacked any of the β-phenethylamines identified in the subgenus Cylindropuntia. All the known alkaloids have been previously isolated from other cacti, but this is the first report of crystalline hordenine from Opuntia. The observed differences between the alkaloid compositions of the two subgenera correlate with their taxonomic divergence. The identification of O-methylated alkaloids, such as mescaline and 3,4-dimethoxy-β-phenethylamine, in the three species O. spinosior, O. imbricata, and O. whipplei supports their classification in a single taxonomic series.