The Opuntia genus, whose origin is in Mexico where several species grow wild, is taxonomically diverse and has become an important crop worldwide. The aim of this work was to assess the molecular composition of five Opuntia species and three different varieties of each taxonomically identified species with different domestication gradients (from the wildest to the most domesticated O. ficus-indica). Data were subjected to chemometric evaluation using principal component analysis (PCA), which showed that O. ficus-indica seems to fall far outside of the current domestication gradient that has been proposed, and its ancestor is still unclear. Phenolic compounds showed major (eucomic acid, kaempferol 3-Omicron-robinobioside-7-Omicron-arabinofuranoside, isorhamnetin 3-Omicron-galactoside, and isorhamnetin 3-Omicron-rhamnoside-7-Omicron-(rhamnosyl-hexoside)) and minor compounds that were present only in wild (kaempferol 3-Omicron-arabinofuranoside) or domesticated (quercetin 3-Omicron-rhamnosyl-(1 -> 2)-[rhamnosyl-(1 -> 6)]-glucoside) species. This information could be very useful for authentication of Opuntia species and the identification of species with the highest potential as sources of compounds with nutritional and therapeutic properties.