We conducted a phylogeographic analysis of the genus Gephyrocharaxin Venezuela to evaluate geomorphologic evidence for the formation of the country's main watersheds and to establish a biogeographical hypothesis of possible diversification mechanisms of the Neotropical freshwater fish fauna. We assayed eight enzyme systems and general proteins to estimate genetic variability (H, P), intraspecific structuring in several Gephyrocharax valencia and G. venezuelae populations (FIS, FIT, and FST), and a phylogenetic approach for the three species of Gephyrocharax in Venezuela, using Corynopoma riisei as the external group. Fourteen presumptive lociindicate that populations of the three species of Gephyrocharaxanalyzed show a clear genetic inter-specific differentiation, determined by four loci with fixed alleles (GPI-B*,IDH*, ME-1*, and ME-2*). The resulting cladogram shows two major clades: a monophyletic group consisting of Gephyrocharax n. sp. and G. venezuelae(restricted to the northwest of the country) and a group formed exclusively by G. valencia (distributed along the largest geographic range). Speciation of the Venezuelan lineages of the genus Gephyrocharaxcould be explained by the origin and course movements of the present Orinoco River together with geomorphologic processes that have occurred in northern Venezuela since the Miocene.