Poecilia vivipara, a small euryhaline guppy is reported at the Maceió River micro-basin in the Fernando de Noronha oceanic archipelago, northeast Brazil. However, the origin (human-mediated or natural dispersal) of this insular population is still controversial. The present study investigates how this population is phylogenetically related to the surrounding continental populations using the cytochrome oxidase I mitochondrial gene from eleven river basins in South America. Our phylogenetic reconstruction showed a clear geographical distribution arrangement of P. vivipara lineages. The Fernando de Noronha haplotype fell within the 'north' clade, closely related to a shared haplotype between the Paraíba do Norte and Potengi basins; the geographically closest continental drainages. Our phylogenetic reconstruction also showed highly divergent lineages, suggesting that P. vivipara may represent a species complex along its wide distribution. Regarding to the insular population, P. vivipara may have been intentionally introduced to the archipelago for the purpose of mosquito larvae control during the occupation of a U.S. military base following World War II. However, given the euryhaline capacity of P. vivipara, a potential scenario of natural (passive or active) dispersal cannot be ruled out.