The rubbernose-pleco genus Chaetostoma comprises 47 currently valid and many undescribed species distributed along Atlantic and Pacific slopes of the Andes Mountains from Panama to southern Peru, the Coastal Mountains of Venezuela, and drainages of the Guiana and Brazilian shields. We present a five-locus molecular phylogeny for 21 described and six undescribed species of Chaetostoma spanning the geographic range of the genus. Bayesian and maximum likelihood analyses found Chaetostoma to be well supported as monophyletic and sister to a clade of central and northern Andean genera that have also been hypothesized to be closely related based on morphology (i.e., Andeancistrus, Cordylancistrus, Dolichancistrus, Leptoancistrus, and Transancistrus). Species of Chaetostoma were divided into a trichotomy consisting of: a Pacific Coast, Central American, Magdalena Basin, Lake Valencia, and Guiana Shield clade a western Orinoco, Lake Maracaibo, and Lake Valencia clade and a widespread upper Amazon/Orinoco clade inclusive of a single species on the Brazilian Shield. We also conducted a systematic review of species from the central Andes of northern Peru and Ecuador. Based on our phylogenetic results and direct examination of historical and recently collected type and non-type material, we describe two new species of Chaetostoma (C. bifurcum, from the Pacific Coast, and C. trimaculineum, from the Atlantic Slope), redescribe four species (C. breve, C. carrioni, C. dermorhynchum, and C. microps, all from the Atlantic Slope), transfer four species from Chaetostoma to Ancistrus and find two species to be junior synonyms.