The South American siluriform fishes are found primarily in the Neotropical region, north and east of the Colorado River of Argentina, with a few relict species distributed southward and westward on both sides of the Andes Mountains. Three of these, the closely related trichomycterids Hatcheria macraei, Trichomycterus areolatus and Bullockia maldonadoi, have been subject to historical taxonomic and nomenclatural arrangements. Here, we amplify a 652-bp fragment of COI mtDNA from 55 H. macraei individuals and use publicly available Cytb mtDNA sequences of the three taxa to assess their relationship, genetic variation and haplotype distribution in relation to hydrographic basins. In addition, we extend a recent morphometric study on H. macraei by analyzing body shape in 447 individuals collected from 24 populations across their entire cis-Andean distribution. We identified some lineages previously assigned to T. areolatus that show a closer relationship to either B. maldonadoi or H. macraei, revealing new boundaries to their currently known trans-Andean distribution. We found a great morphologic variation among H. macraei populations and a high genetic variation in H. macraei, T. areolatus and B. maldonadoi associated with river basins. We highlight further integrative studies are needed to enhance our knowledge of the southern Andean trichomycterid diversity.