Background and aims – Endemism may be defined according to the time of origin of taxa. Neo-endemics refer to relatively recent species that have not dispersed outside their ancestral areas. In contrast, paleo-endemics refer to species of ancient origins, which are currently geographically restricted but probably were more widespread in the past. Geographically, endemism areas may also be based on the co-occurrence of more than one species. We aimed to qualitatively identify the neo-endemism and paleo-endemism of endemic Cactaceae of the Tehuacán-Cuicatlán Valley, as well as to quantitatively assess paleo- and neo-endemics areas. Material and methods – Using a dated molecular phylogeny of endemic Cactaceae, we defined paleo- and neo-endemics using an arbitrary boundary of 2.6 million years ago; we also assessed the significance of concentrations of these species using a categorical analysis of paleo- and neo-endemism. Key results – Our results showed that most endemic Cactaceae in the Tehuacán-Cuicatlán Valley arose throughout the Pleistocene, while categorical analysis indicated localised mixed- and super-endemism (including both paleo- and neo-endemics) areas. Conclusion – We suggest that paleo- and neo-endemics, as well as localised mixed-endemism areas, may have originated due to a probable high climatic stability in the Tehuacán-Cuicatlán Valley, which in addition to topographically rugged and ecologically complex zones (e.g. ecotones, isolated habitat patches) may have allowed it to function as a refuge throughout Pleistocene climatic changes, mainly promoting the speciation of neo-endemics, as well as the persistence of relatively few paleo-endemics.