Trichomycterus has been considered for a long time to be the most problematic genus of the diverse neotropical subfamily Trichomycterinae. Recently, Trichomycterus was restricted to a clade from eastern South America supported by molecular data, but no unique morphological character state was found to distinguish it, making it difficult to allocate new species based on morphology alone. The objectives of this study were to conduct an osteological comparative analysis comprising a large sample of valid species of Trichomycterus, to conduct a total evidence phylogenetic analysis, combining osteological characters and a multigene database, and to propose an intrageneric classification based on the results of the phylogenetic analysis. Fifty-two osteological characters were combined with a multigene molecular data set of 2974 bp for 44 species of Trichomycterus, and 21 outgroups generated a well-supported phylogenetic tree, making it possible to delimit and diagnose intrageneric lineages, of which six subgenera are recognized. The high morphological diversity of osteological structures herein first reported for Trichomycterus from eastern South America is possibly related to some ecological specializations. This study shows that osteological characters combined with molecular data may be useful to consistently delimit and distinguish between trichomycterines, shedding light on the still persistent problems in trichomycterine systematics.