The giant Mediterranean limpet Patella ferruginea Gmelin, 1791 is an endangered marine gastropod, whose range has progressively shrinked to few, restricted areas, due to intense human exploitation. We have studied the genetic structure of the species, in order to (1) gather information about the levels of genetic variability within and between natural populations of P. ferruginea collected in two Sardinian marine protected areas (MPAs) (Penisola del Sinis—Isola di Mal di Ventre and Isola dell' Asinara), and (2) make an attempt to find relationships between ecological and biological attributes of the species and the genetic differentiation of the populations studied. The genetic study was carried out by means of the analysis of ten inter-simple sequence repeat (ISSR) primers on a total of 40 individuals collected at four localities. Genetic analysis evidenced (1) medium to high levels of within-population genetic variability, (2) a pattern of genetic structuring that varied with spatial scales, and (3) a strong genetic differentiation between the two MPAs. Although preliminary, these results suggest that gene flow may be present only at very small geographic scale, raising concerns on the future of the conservation of the species.