The genetic structure and population connectivity of the Mediterranean endemic speckled skate Raja polystigma were investigated in 10 population samples (N = 232) at 7 exon-primed nuclear microsatellites and at 3 mitochondrial DNA sequence markers. The phylogeographic and population genetic analyses revealed that R. polystigma in the western and central Mediterranean represents a near-panmictic population, with a subtle but significant mitochondrial divergence of the Adriatic deme. Nuclear genotypes revealed that 2.5% of the total individuals exhibited an admixed ancestry with the sibling species R. montagui (spotted ray). Individuals with admixed ancestry were detected along with purebred individuals in the Algerian, southern Tyrrhenian, Sicilian and Adriatic R. polystigma population samples, but they were absent or rare in Sardinian and northern Tyrrhenian ones. Since the 2 species co-occur in the southwestern Mediterranean, we suggested that this area may act as a secondary hybrid zone.