The phylogenetic relationships of Pachycereus (Cactaceae) species and relatives from subtribe Pachycereinae were studied using DNA sequence data. The plastid rpl16 intron, trnL intron, trnL-F intergenic spacer, and nuclear rDNA internal transcribed spacer region (ITS) were sequenced for 30 species, representing the four genera of subtribe Pachycereinae (Carnegiea, Cephalocereus, Neobuxbaumia, and Pachycereus) as well as three additional outgroup genera from subtribe Stenocereinae. Phylogenetic analyses support neither the monophyly of Pachycereus as currently circumscribed nor Pachycereinae unless Stenocereus aragonii and S. eichlamii are included within it. However, these results suggest that the subtribe can be divided into three major clades. The first includes Pachycereus hollianus and P. lepidanthus, which is sister to a large clade combining species from the Pachycereus and Cephalocereus groups. Within this large clade Cephalocereus and Neobuxbaumia together with Pachycereus fulviceps are sister to the remaining species of Pachycereus as well as Stenocereus aragonii, S. eichlamii, and Carnegiea gigantea. Our results suggest that Pachycereus is paraphyletic and that several other genera (Backebergia, Lemaireocereus, Lophocereus, and Pseudomitrocereus) may be resurrected to accommodate these new phylogenetic insights. A number of morphological and anatomical characters support these relationships, indicating that future analyses combining both molecular and morphological characters will be particularly useful in resolving relationships within this group of columnar cacti.