Accurate species delimitation is crucial for studies of phylogeny, phylogeography, ecology, conservation and biogeography. The limits of species and genera in the Characidae family are controversial due to its uncertain phylogenetic relationships, high level of morphological homoplasy and the use of ambiguous morphological characters for descriptions. Here we establish species boundaries for Bryconamericus, Hemibrycon, Knodus and Eretmobrycon (Stevardiinae: Characidae), previously diagnosed with morphology, using three different barcoding approaches (GMYC, PTP, ABGD). Results revealed that species delimitation was successful by the use of a single-gene approach and by following a workflow in the context of integrative taxonomy, making evident problems and mistakes in the cataloging of Characidae species. Hence, it was possible to infer boundaries at genus level for clusters in the trees (GMYC and PTP) and automatic partitions (ABGD) which were consistent with some of recent taxonomic changes proposed in Characidae. We found that discordance cases between methods were linked to limitations of the methods and associated to putative species cluster closely related, some historically problematic in their diagnosis and identification. Furthermore, we suggested taxonomic changes and possibly new species, revealing a high degree of hidden diversity. Finally, we propose a workflow as a fast, accurate and objective way to delimit species from mitochondrial DNA sequences and to help clarify the classification of this group.