The genus Pseudancistrus is widely distributed in rocky, fast-flowing habitats of rivers draining all sides of the Guiana Shield and the northern slope of the Brazilian Shield. Initially diagnosed as having an “interoperculum” weakly evertible and lacking elongate odontodes, the definition of Pseudancistrus was subsequently expanded to include species with long, evertible cheek odontodes. Within Pseudancistrus sensu lato were included the already available genera Guya­nancistrus and Lithoxancistrus. To evaluate the relevance of such a grouping, and particularly the morphology-based hypotheses of Armbruster (2004a, 2008), we reconstructed a phylogeny using mitochondrial and nuclear data and samples from 13 species currently included in Pseudancistrus sensu lato. All phylogenetic reconstructions recovered Pseudancistrus sensu lato as a paraphyletic assemblage of five unrelated lineages, and most of the alternative hypotheses evaluating monophyly of the genus were significantly rejected. Two of the recovered lineages were consistent with the genera Guya­nancistrus and Lithoxancistrus, which are accordingly revalidated. A fourth lineage consisted of Pseudancistrus pectegenitor and P. sidereus, which may represent an undescribed genus. However their placement within Lithoxancistrus cannot be excluded. The fifth lineage consisted only of P. genisetiger, which was recovered as sister to Hemipsilichthys gobio and may represent an undescribed genus within the Delturinae.