Since the first authorised introductions to Spain in 1921 of the Eastern mosquitofish Gambusia holbrooki (Girard, 1859), this North American freshwater fish species has invaded large parts of several Iberian basins. For the River Ebro basin, we report 24 new records of this exotic species between 2005 and 2007 which represents a considerable expansion. Potential negative implications for aquatic invertebrate and fish communities are discussed.