Eight microsatellite markers were used to examine the historical and contemporary factors influencing the distribution of genetic variation within and among populations of Fundulus diaphanus (Lesueur, 1817) in the St. Lawrence River. Our results show that the contemporary hydrodynamics of the river affect levels of diversity and differentiation in this species. Genetic diversity increased towards downstream sites and levelled off at the upstream end of the tidal section, in the vicinity of Lake St-Pierre outlet. Likewise, differentiation and isolation-by-distance were most pronounced in the upstream, strictly fluvial section of the river. Surprisingly, however, we did not detect any significant intershore differentiation in this riparian fish species. Historical influence was also clearly detected and transcended the contemporary pattern, as revealed by an extremely strong isolation-by-distance pattern along the entire river stretch. This pattern likely results from the historical, postglacial secondary contact between two glacial races, as indicated by the clinal variation of constrasting allele size frequency distributions along the river and the significantly greater values of differentiation indices considering mutational information.