Three distinct and independent molecular-based species delimitation analyses were performed among the species and populations included within theAustraloherosautranigroup, based on sequences of the mitochondrial gene Cytochrome b: a tree-based method proposed by Wiens and Penkrot (WP), a Character-based DNA Barcoding (CBB) and coalescent species delimitation method termed the Bayesian Implementation of the Poisson tree processes (bPTP). The congruence of WP and CBB delimited 11 independent lineages (species), while the bPTP delimited just nine lineages. We did not favour any of the methods, and we considered the possibility of two slightly variant scenarios. A time-calibrated phylogenetic analysis is proposed based on the predominant congruence of the results of these three species delimitation methods herein applied. The monophyly of theA.autranispecies group was highly supported with maximum node support value and diagnosed by 11 nucleotide substitutions. The sister clade of theA.autranispecies group is the clade comprisingA.sp. Timbé do Sul andA.minuano. The phylogenetic analysis supports three main clades within theA.autranispecies group, supported by maximum node support value, with the Southern Mata Atlântica clade as the most basal clade. Divergence time estimates indicate that the diversification of theAustraloherosoriginated during the early Neogene, but only in the late Neogene did the processes of diversification in the southeast and north regions occur. Diversification within theAustraloherosautranispecies group occurred synchronically for the three main clades during the beginning of the Quaternary. It is demonstrated that molecular characters are valuable tools for species recognition, particularly in speciose groups with inconspicuous or difficult to record morphological characters. The resulting phylogeny of theAustraloherosautranigroup is highly compatible with the geological and biogeographic scenarios proposed for the Neogene and Quarternary shaping of the extant river basins of eastern Brazil. Despite the origin of theA.autranigroup being dated to the late Miocene, species level diversification occurred in the Pleistocene and was probably driven by headwater capture events and sea-level fluctuations.