Posidonia oceanica is an endemic seagrass species in the Mediterranean Sea. In order to assess levels of genetic structure in this species, the microsatellite polymorphism was analysed from meadows collected in several localities, along the coasts of the Tyrrhenian Sea (Mediterranean Sea). The existence of single population units and the recruitment of seedlings collected in some localities were investigated. Moreover, genetic structure at different spatial scales and biogeographic relationships among populations were also assessed. Our analysis showed the existence of clear patterns of genetic structure in P. oceanica in the area considered in the analysis. P. oceanica, in fact, is present in separate meadows that represent discrete populations, characterized by low genetic diversity. Comparable levels of genetic variability between mature meadows and seedlings were found. Patterns of genetic relatedness among populations seem to be in accord with direction of dominant current flux in the whole area, separating South Tyrrhenian from North Tyrrhenian populations. Moderate levels of gene flow between populations and genetic substructure within populations, together with the finding of the limited role of sexual reproduction in increasing genetic variability, should be a cause for concern for the persistence of this essential resource in the Mediterranean basin.