﻿AbstractPhenacogasteris the most species-rich genus of the subfamilyCharacinaewith 23 valid species broadly distributed in riverine systems of South America. Despite the taxonomic diversity of the genus, little has been advanced about its molecular diversity. A recent molecular phylogeny indicated the presence of undescribed species withinPhenacogasterthat is formally described here. We sampled 73 specimens ofPhenacogasterand sequenced the mitochondrial cytochrome c oxidase subunit I (COI) gene in order to undertake species delimitation analyses and evaluate their intra- and interspecific genetic diversity. The results show the presence of 14 species, 13 of which are valid and one undescribed. The new species is known from the tributaries of the Xingu basin, the Rio das Mortes of the Araguaia basin, and the Rio Teles Pires of the Tapajós basin. It is distinguished by the incomplete lateral line, position of the humeral blotch near the pseudotympanum, and shape of the caudal-peduncle blotch. Meristic data and genetic differentiation relative to otherPhenacogasterspecies represent strong evidence for the recognition of the new species and highlight the occurrence of an additional lineage ofP.franciscoensis.