[Background] Marine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multilocus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina).[Results] We investigated genetic structure of sardine among nine locations in the Atlantic Ocean and Mediterranean Sea using allelic size variation of eight specific microsatellite loci. Bayesian clustering and assignment tests, maximum likelihood estimates of migration rates, as well as classical genetic-variance-based methods (hierarchical AMOVA test and RST pairwise comparisons) supported a single evolutionary unit for sardines. These analyses only detected weak but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test.[Conclusion] We suggest that the discordant genetic structuring patterns inferred based on mitochondrial and microsatellite data might indicate that the two different classes of molecular markers may be reflecting different and complementary aspects of the evolutionary history of sardine. Mitochondrial data might be reflecting past isolation of sardine populations into two distinct groupings during Pleistocene whereas microsatellite data reveal the existence of present day gene flow among populations, and a pattern of isolation by distance.This work received financial support from a project of the MEC (Ministerio de Educación y Ciencia) to R. Z. (REN 2001-1514/GLO) and AECI-MAE (Agencia Española de Cooperación Internacional – Ministerio de Asuntos Exteriores) project to T. Atarhouch and R.Z (project n° 168/03/P). E.G.G. was sponsored by a predoctoral fellowship of the MEC.Peer reviewed