The marine environment is characterized by few physical barriers, and pelagic fishes commonly show high migratory potential and low, albeit in some cases statistically significant, levels of genetic divergence in neutral genetic marker analyses. However, it is not clear whether low levels of differentiation reflect spatially separated populations experiencing gene flow or shallow population histories coupled with limited random genetic drift in large, demographically isolated populations undergoing independent evolutionary processes. Using information for nine microsatellite loci in a total of 1951 fish, we analyzed genetic differentiation among Atlantic herring from eleven spawning locations distributed along a longitudinal gradient from the North Sea to the Western Baltic. Overall genetic differentiation was low (θ = 0.008) but statistically significant. The area is characterized by a dramatic shift in hydrography from the highly saline and temperature stable North Sea to the brackish Baltic Sea, where temperatures show high annual variation. We used two different methods, a novel computational geometric approach and partial Mantel correlation analysis coupled with detailed environmental information from spawning locations to show that patterns of reproductive isolation covaried with salinity differences among spawning locations, independent of their geographical distance. We show that reproductive isolation can be maintained in marine fish populations exhibiting substantial mixing during larval and adult life stages. Analyses incorporating genetic, spatial, and environmental parameters indicated that isolating mechanisms are associated with the specific salinity conditions on spawning locations.