The occurrence of genetic differentiation among western Mediterranean Hediste diversicolor (Polychaeta: Nereididae) populations was assessed by allozyme electrophoresis on cellulose acetate. Seventeen loci were analysed in four populations. The level of the genetic variability was markedly low (mean HL range: 0.014–0.034), but comparable to that of other brackish water nereidids. The values of Nei genetic distance index (D) confirm the existence of genetic differentiation between the geographically isolated populations at Venice, Elba, Navicelli and Serchio D range: 0.128–0.356). However, the two samples from Serchio and Navicelli, ~15 km apart, were not genetically different (D=0·00005). The level of genetic differentiation in H. diversicolor populations followed the isolation-by-distance model. Reduced gene flow among H. diversicolor populations may be explained by its limited dispersal capacity and the eco-physiological barriers that occur between different brackish habitats.