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The Pleistocene glacial period shaped the current genetic structure of numerous species. The last glacial dynamics has been proposed to have split the South American sea lion, Otaria flavescens , into two Evolutionarily Significant Units (ESUs), one on each side of the continent. However, previous studies have not provided genetic information on colonies found along 3000 km of coastline of the southernmost limit of the species distribution, where gene flow could occur. We conducted an exhaustive phylogeographical analysis of O. flavescens using a mtDNA marker, including, for the first time, data from colonies living south of latitude 45° S, in the Argentinian provinces of Santa Cruz and Tierra del Fuego. Our results indicated the presence of five Conservation Units across the distribution range of O. flavescens and suggest that the Patagonian population must have expanded about 150 000 BP. We found evidence for gene flow across the entire species range, supporting a scenario of secondary contact in Tierra del Fuego where representatives of the oldest lineages coexist. The presence of gene flow between oceans leads us to reject the assumption of complete reciprocal monophyly for mtDNA between the presumed ESUs, suggesting that the species constitutes a single Evolutionarily Significant Unit. [ABSTRACT FROM AUTHOR]