The catfish family Heptapteridae is ubiquitous across a range of freshwater habitats from southern Mexico to northern Argentina and contains 23 genera and 228 valid species. After a century of mostly morphology-based systematic analyses of these fishes, we provide the first molecular phylogenetic hypothesis spanning most valid Heptapteridae genera (16 of 23). We examined eight of 14 valid genera in the Nemuroglanis-subclade (Heptapterini), all valid genera in the Brachyglanis-subclade (Brachyglaniini) and most valid Brachyglaniini species (11 of 15). Maximum likelihood and Bayesian analyses of a 4156-base alignment of five gene regions (three mitochondrial: COI, Cyt b, and ND2; two nuclear: RAG2, Glyt) yielded thoroughly resolved and statistically robust phylogenies that were largely congruent with each other and with previous morphology-based hypotheses. We propose a revised phylogenetic classification consisting of two subfamilies (Rhamdiinae, Heptapterinae) each with two tribes. Dense taxonomic sampling of Brachyglaniini, including type species of Brachyglanis, Gladioglanis, Leptorhamdia, and Myoglanis, revealed widespread paraphyly. Newly recovered clades within Brachyglaniini are closely associated with either the upper Orinoco or the Essequibo suggesting repeated dispersals and/or range expansions/contractions across the western Guiana Shield highlands and from there to the upper Amazon and Brazilian Shield. These biogeographical processes appear to have been an important driver of allopatric diversification in the clade.