An analysis of chloroplast DNA restriction site variation was undertaken to investigate the evolutionary divergence of Ferocactus and its possible relationship with North American columnar cacti of tribe Pachycereeae. Our chloroplast DNA study using parsimony-based phylogenetic reconstruction methods provides molecular synapomorphies to define major lineages within Ferocactus and columnar cacti of the tribe Pachycereeae. The issue of monophyly in Ferocactus remains problematic: it appears to be a paraphyletic assemblage derived from an Echinocactus-like ancestor from which three major lineages have evolved. Also, there is a lack of support for the hypothesized basal position of. F. flavovirens and F. robustus, and no evidence was found to favor the phylogenetic relationship of Ferocactus with North American columnar cacti, in particular with Escontria chiotilla. For the columnar cacti, the study supports the monophyly of the tribe Pachycereeae as well as its two subtribes: Stenocereinae and Pachycereinae. Ln the Stenocereinae, Stenocereus dumortieri was found to be in a basal position, and forms a phylogenetically distinct lineage from Stenocereus s. sh. and other columnar cacti such as Escontria, Polaskia, and Myrtillocactus. This supports the segregation of S. dumortieri from Stenocereus, and the resurrection of Isoiatocereus dumortieri as a distinct genus.