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Aim Limnomysis benedeni Czerniavsky, 1882 is a mysid crustacean native to the Ponto-Caspian (Black and Caspian Sea) rivers and estuaries, and has recently spread across Europe through intentional and unintentional introductions. We explored the structuring of genetic variation in native and non-native populations with an aim to trace the sources of the invasions, and to infer whether the spread has occurred through a single or multiple invasion waves. Location Native estuaries in the Ponto-Caspian basin (Volga, Don, Dnieper, Dniester, Danube) and the recently colonized range along the Danube–Rhine river systems and Lithuania. Methods A fragment of the mitochondrial COI gene was sequenced to assess genetic affinities and diversity in native and recently established populations. Results The genetic diversity in the native regions is organized into several strongly diverged haplotype groups or lineages, partly allopatric, partly sympatric. All these lineages have also spread beyond the native range. Even the recent rapid dispersal across Europe along the Danube–Rhine system towards the North Sea basin involved several lineages from the Danube delta sector. The structuring of genetic diversity among invaded sites suggests multiple invasion events to the Danube–Rhine drainage. This contrasts with data from some other Ponto-Caspian species, where a single haplotype seems to have occupied most invaded areas. There is no evidence that intentionally stocked reservoirs in the Baltic Sea basin would have contributed to further unintentional spread of L. benedeni. Main conclusions Limnomysis benedeni is spreading across Europe using the southern invasion corridor. The invasion most likely involved several waves from differentiated sources in the native Danube delta area. [ABSTRACT FROM AUTHOR] more...