(1) Background: Trichomycterinae represent 60% of the species in the family and, while seven genera comprise 1–3 species each, Trichomycterus and Cambeva have over 180 known species between them. Although integrative studies aimed to clarify the relationships within the subfamily, the diversity of species of Trichomycterus remains an open question. Herein, we explored an unprecedented sample to investigate the divergence in the lineages of Trichomycterus. (2) Methods: we recovered the phylogenetic relationships of the subfamily using 566 sequences (999 bp) of the mitochondrial gene cytochrome b, calculated intra- and intergroup distance percentages, and estimated divergence times. (3) Results: we recovered 13 highly supported and geographically structured lineages; intergenus divergence was 11–20%, while interspecies divergence was 3–11%; Trichomycterus, Cambeva, Scleronema, Hatcheria, Eremophilus, and Ituglanis were recovered as monophyletic, with three other highly divergent clades: Guiana Shield, Magdalena basin, and Tapajós basin. (4) Conclusions: We propose that the trans-Andean austral clades be allocated into Hatcheria, and the Guiana clade supports a new genus. We also observed that the headwaters nearest the Magdalena and Orinoco basins showed a high diversity and endemism of Trichomycterinae lineages. We discussed the role of geomorphological events and the climatic features which may explain cladogenesis events in Trichomycterinae.