A new species of Eigenmannia is described from the Rio Branco basin, Roraima, Brazil, based on morphological and molecular datasets. It is distinguished from all congeners by the following combination of characters: lateral line stripe extending from first perforated lateral line scale to distal portion of caudal filament, presence of superior midlateral stripe with origin posterior to end of body cavity anal‐fin hyaline, caudal filament corresponding to 15.2%–43.1% LEA, subterminal mouth, ii,14–16 pectoral‐fin rays, 166–219 anal‐fin rays, 10–13 scale rows above lateral line at vertical through posterior tip of pectoral fin, 100–128 scales on lateral line, 22–28 premaxillary teeth, 19–23 dentary teeth, 7–10 endopterygoid teeth, depth of posterodorsal expansion on infraorbitals 1 + 2 half as long as infraorbitals 1 + 2 length, basibranchial 1 unossified, 13 precaudal vertebrae, and length of coronomeckelian bone corresponding to 20% of Meckel's cartilage length. The new species has significant genetic divergence from species with accessible DNA sequences in public repositories, ranging from 10.8% to 17.7%. An osteological description of the new species, a review of Eigenmannia cytochrome c oxidase subunit I (COI) sequences available in public repositories based on voucher examination, and a hypothesis of phylogenetic relationships for the new species based on COI are provided. The critical importance of including voucher examination as one of the steps in the pipeline for using DNA sequences present in public repositories in taxonomic and phylogenetic studies is discussed.