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Poeciliopsis (Cyprinodontiformes: Poeciliidae) is a genus comprised of 25 species of freshwater fishes. Several well-known taxonomic uncertainties exist within the genus, especially in relation to the taxonomic status of Poeciliopsis pleurospilus and P . gracilis . However, to date, no studies have been conducted to specifically address the taxonomic status of these two species. The goal of this study was to examine the taxonomic validity of P . pleurospilus and P . gracilis using genomic data (ddRADseq) in phylogenetic, population genetic, and species delimitation frameworks. Multiple analyses support the recognition of both taxa as distinct species and also permits us to revise their respective distributions. A species delimitation analysis indicates that P . pleurospilus and P . gracilis are distinct species, each of which consists of two distinct lineages that are geographically structured. Phylogenetic and population genetic analyses provide clear evidence that individuals of P . gracilis are distributed north and west of the Isthmus of Tehuantepec in both Pacific and Atlantic river systems in Mexico, whereas individuals of P . pleurospilus are distributed in both Atlantic and Pacific river systems south and east of the Isthmus of Tehuantepec, from southern Mexico to Honduras.