Neotropical freshwaters host more than 4,000 fish species, of which almost one quarter are suckermouth armored catfishes of the family Loricariidae – the largest catfish family and fifth most species-rich vertebrate family on Earth. Given their diversity and ubiquitous distribution across many habitat types, loricariids are an excellent system in which to investigate factors that create and maintain Neotropical fish diversity, yet robust phylogenies needed to support such ecological and evolutionary studies are lacking. We seek to buttress the systematic understanding of loricariid catfishes by generating a genome-scale data set (1,041 loci, 328,330 bp) for 140 species spanning 75 genera and five of six previously proposed subfamilies. Both maximum likelihood and Bayesian analyses strongly supported monophyly of Loricariidae, with early diversification dated to Late Cretaceous at ~65 millions of years ago. Our results also corroborate monophyly and previously hypothesized relationships among all examined subfamilies: Delturinae was sister to all other analyzed loricariids, with subfamily Rhinelepinae diverging next, followed by Loricariinae sister to Hypostominae + Hypoptopomatinae. These results reinforce the established backbone of loricariid interrelationships and lay a strong foundation for future research to focus on intergeneric relationships within subfamilies.