A new species of the loricariid genus Lithogenes Eigenmann, 1909, is described on the basis of 84 specimens captured from a single locality in the upper Río Orinoco drainage of southern Venezuela. The new species is only the third representative of the subfamily Lithogeninae to be recognized in the 100 years since the discovery of the type species, L. villosus, and is the only lithogenine known from more than a handful of specimens. This new material provides the basis for a comprehensive review of lithogenine systematics, comparative anatomy, and interrelationships. Lithogenes wahari, new species, shares with its congeners the dermal plates of the trunk comprised of three paired series, presence of a bifurcate levator arcus palatini crest and expanded lateral lamina of the hyomandibula, and the palatine sesamoid not reaching the nasal capsule, thus confirming its placement in Lithogenes among the Loricariidae. The new species is diagnosed among congeners by the absence of odontodes on the proximal portion of the ventral surface of the first pelvic-fin ray (vs. ventral pad covered with embedded odontodes along entire length) and thickened skin of the pelvic pad forming extensive ridges; accessory premaxillary teeth absent; anal fin with intense pigment band along base and diffuse spot at midlength of fin rays (vs. pigment band at base absent, fin rays dusky, without distinct spot). Characters useful for distinguishing lithogenine species are reviewed; revised diagnoses and descriptions are provided for the two previously described species in light of new character evidence. A detailed comparative analysis of the osteology and myology of L. wahari is presented and discussed relative to homologous conditions observed more broadly among the loricarioid catfishes. Of particular importance are aspects of musculoskeletal anatomy that are hitherto unknown for lithogenines, and aspects of sexual dimorphism and the anatomy of the reproductive and digestive systems that are unique or unusual among loricariid catfishes. A phylogenetic analysis of relationships among species based on morphological characters places the two Guyana Shield species (L. villosus and L.wahari) as sister taxa on the basis of four synapomorphies. Both species share reduction in the width and extent of the jaws, resulting in the derived reduction in the numbers of teeth carried by the jaw elements. Evaluated with respect to the geographic distribution of the species, the pattern of phylogenetic relationships suggests an ancestral widespread distribution for the Lithogeninae throughout the Guyana Shield plus the Caribbean and eastern Andean foreland basin of northern South America, followed by vicariance and subsequent divergence of populations now isolated in the coastal mountains of northern Venezuela and the Guyana Shield region. Lithogenine catfishes share a number of unique features with astroblepid catfishes that are not observed to occur in other members of the Loricariidae, such as the morphology of the pelvic fins, specialized pelvic musculature, and associated adaptations for climbing. Evaluated against the evidence supporting their phylogenetic placement as the sister group to all other Loricariidae, exclusive of the Astroblepidae, these shared similarities suggest that the association with rocky habitats of headwater stream systems and the ability to climb vertical surfaces may represent ancestral conditions for the lineage leading to the astroblepid plus loricariid catfishes.