The brown trout (Salmo trutta L.) is widely distributed all around Europe but its natural diversity is threatened by massive stocking with Atlantic domestic strains. Describing the remaining natural genetic diversity and the proportion of domestic hatchery strains in rivers is a prerequisite for smart conservation. The high genetic diversity of brown trout populations around the Tyrrhenian Sea is well known. Use of twelve microsatellites has allowed description of the natural genetic structure of populations and detection of the consequences of stocking. Mitochondrial DNA control region sequences and the LDH-C1* gene enabled placement of each population into one of the six mitochondrial and two allozymic known evolutionary lineages. The Corsican populations showed low intra-population genetic diversity but an exceptionally high level of inter-population differentiation. More southern Tyrrhenian regions exhibited opposite pattern of diversity, partly due to the Atlantic domestic introgression. Globally, the natural structure outlines two north–south clines: high inter-population differentiation and predominance of the Adriatic lineage in the north, but lower inter-population differentiation and the presence of the natural Atlantic lineage in the south. In addition, the Tyrrhenian region is the contact zone between the widespread Adriatic lineage and a local natural Atlantic lineage probably coming from North Africa through the Strait of Gibraltar.