A phylogenetic analysis of nearly all genera of the Hypostominae and the Ancistrinae is provided based on osteology, external anatomy, and digestive tract anatomy. The results suggest that the Hypostominae is a paraphyletic assemblage. Delturus and Upsilodus form a monophyletic group sister to all other loricariids. Hemipsilichthys, Isbrueckerichthys, Kronichthys, and Pareiorhina form a monophyletic group with Neoplecostomus and the Hypoptopomatinae and are transferred to the Neoplecostominae. The remainder of the Hypostominae is made paraphyletic by the continuing recognition of the Ancistrinae. Ancistrinae is returned to the Hypostominae and recognized as a tribe, Ancistrini. In addition, four new tribes (Corymbophanini, Hypostomini, Pterygoplichthini, and Rhinelepini) are described. Hypostomus is also paraphyletic, the bulk of it forming a monophyletic clade with Aphanotorulus, Cochliodon, and Isorineloricaria. All of the potential monophyletic groups within Hypostomus grade into one another; therefore, Aphanotorulus, Cochliodon, and Isorineloricaria are placed in the synonymy of Hypostomus. Pterygoplichthys and Glyptoperichthys are also polyphyletic, and Liposarcus and Glyptoperichthys are recognized as synonyms of Pterygoplichthys. Sister to Pterygoplichthys is the Hemiancistrus annectens group (including Hypostomus panamensis) which represents an undescribed genus. The phylogeny presented is compared with previous hypotheses. © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society, 2004, 141, 1−80.