Echinocereus palmeri subsp. mazapil, a new subspecies of Cactaceae from the Chihuahuan Desert Region, Mexico, is described and illustrated. It has only been collected from a highly specialized habitat at two remote localities in the state of Zacatecas and its range is markedly disjunct from that of the typical subspecies. In addition to divergences in geographic range and habitat preferences, E. palmeri subsp. mazapil differs from the typical subspecies by usually having relatively smaller stems with tuberculate ribs and less numerous radial spines, which usually are of unequal length.