The genus Pachycereus is restricted to Guatemala, Mexico, and the SW United States and includes 13 species according to the most recent classification. A phylogenetic analysis was conducted in order to test the monophyly of Pachycereus with respect to other genera of subtribe Pachycereinae. A matrix of 44 structural characters (morphology and anatomy) was scored for 29 taxa, including nine species of the Pachycereinae. Results obtained indicated that the subtribe Pachycereinae can only be monophyletic if two species of Stenocereus (S. aragonii and S. eichlamii) are included in it, based on two seed characters (smooth periclinal wall and finely rough relief). Pachycereus is not monophyletic according to the most recent classification. Pachycereus hollianus and P. lepidanthus lack a differentiated fertile zone, and form a basal group in the subtribe Pachycereinae; they do not belong to the genus Pachycereus. Pachycereus fulviceps is related to the genera Cephalocereus and Neobuxbaumia based on nectar zone type, color of fruit pulp, and prismatic calcium oxalate crystals in the epidermic cells. Pachycereus grandis, P. pectenaboriginum, P. pringlei, P. tepamo, and P. weberi are a monophyletic group, based on the presence of a narrow interareolar groove and flowers with a basally constricted tube. This group of five species comprises the genus Pachycereus sensu stricto.