Pejerrey is the common name given to Odontesthes species from South America. Every year, individuals of pejerrey called “Gran (Big) Parana” appear during the low-temperature season at rivers in the southern section of the La Plata Basin. Gran Parana fishes are highly appreciated for fishing, and present some biological features different from other well-characterized Odontesthes fish, such as bigger size and migratory behavior. Regulations for the management of pejerrey fisheries within La Plata Basin have not been implemented yet. The aims of the present work were to characterize the Gran Parana pejerrey species by molecular methods and carry out the first population genetic study of Gran Parana pejerrey from the La Plata Basin. All Gran Parana specimens were classified as O. bonariensis, based on both morphology and microsatellite loci. Genetic differentiation was observed between Gran Parana pejerrey and O. bonariensis pejerrey sampled at Chascomus lagoon. In addition, temporal genetic differentiation was observed, suggesting the presence of different cohorts migrating along the basin. The knowledge of population dynamics and differentiation will contribute to figure out fisheries models and to design protective areas for this species under commercial exploitation.