| Summary: | <i>Hypomasticus copelandii</i> is a Neotropical freshwater fish widely distributed across coastal drainages of southeastern Brazil, a highly impacted region of South America. The interspecific phylogenetic relationships within the genus and the taxonomic status of the species remain uncertain. Using two mitochondrial and one nuclear locus, we performed a phylogenetic, species delimitation, and time-calibrated analyses to test the hypothesis that <i>H. copelandii</i> is a species complex currently delimited by different Atlantic coastal systems. Results indicate that <i>H. copelandii</i> presents two well-delimited genetic lineages: one in the northern drainages of the Jucuruçu, Mucuri and Doce rivers, and the other in the southern region represented by the Paraíba do Sul River Basin. The time-calibrated phylogeny indicated a split between the two genetic lineages at around 2.8 million years ago (Ma), which might be related to headwater capture events during the Plio-Pleistocene. The discovery of a distinct genetic lineage for <i>H. copelandii</i> suggests distinct management plans for the northern and southern drainages. Such hidden diversity within the <i>H. copelandii</i> provides useful information for taxonomy and conservation across a severely impacted region of Brazil.
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