The Rio Doce basin is situated in a biodiversity hotspot, with some fish groups still superficially known. The intense process of habitat loss and water contamination justifies efforts to speed up the pace of describing fish species diversity. Herein, we focus on a clade of trichomycterine catfishes, subgenus Psammocambeva, genus Trichomycterus, performing a multigene phylogenetic analysis, which integrated to a comparative morphological analysis corroborates monophyly and relationships of four species complexes. Five new species are recognised and described. Species distribution patterns support the recognition of five areas of endemism for trichomycterines, most of which are well-known biodiversity centres: Serra do Espinhaço, Serra do Brigadeiro, Serra do Caparaó, Serra do Castelo, and upper-middle Rio Doce. Trichomycterine diversity in the Rio Doce basin, with 16 species, of which 15 belong to Psammocambeva, is lower than in the smaller neighbouring Rio Paraíba do Sul basin, with 21 species in five subgenera. Data suggest that species diversification in the former basin is relatively younger, following the initial diversification of Psammocambeva in the Miocene, whereas diversification in the latter basin has been estimated to have occurred during the Oligocene. This study reinforces the importance of naming new taxa and delimitating areas of endemism as primary tools for conservation strategies.