Callichthys serralabium, a new callichthyid catfish species, is described from drainages in the upper Orinoco River, near La Esmeralda, Venezuela and the headwaters of the Negro River, tributary of the Amazon River, both in Brazil and Venezuela. This species is readily distinguished from other Callichthys by possessing a serrated lower lip, 8–9 branched rays in the pectoral fin, an irregular color pattern of dark, diffuse blotches on flanks of adults, a longer anal-fin spine, and absence of the prenasal central plate. A key for the species of Callichthys is provided and some characters incongruent with the current phylogenetic hypothesis for the callichthyids are discussed.