The first records of Platanichthys platana in the upper Rio Paraná floodplain (URPF) date to 2012, but variations in the body length of specimens collected from this basin have since generated uncertainty about their identity and origin of introduction. This study aimed to genetically characterize populations of P. platana in different river basins, assess haplotype diversity, and determine the origin of the introduced population in the URPF. Sequences from the COI region were used to calculate genetic distances, construct gene trees, and identify haplotype networks. Eight haplotypes were found, several of which were exclusive to the coastal basins of Santa Catarina and Rio Grande do Sul, and the Río Negro basin (Río Yí). The Río de La Plata and Mar Chiquita basins, and upper Rio Paraná floodplain shared haplotypes. Based on the results of our analysis, we concluded that the URPF specimens were introduced from populations in Argentina.