A major goal of population genetics research is to identify the relative influences of historical and contemporary processes that serve to structure genetic variation. Most population genetic models assume that populations exist in a state of migration-drift equilibrium. However, in the past this assumption has rarely been verified, and is likely rarely achieved in natural populations. We assessed the equilibrium status at both local and regional scales of the Atlantic killifish, Fundulus heteroclitus. This species is a model organism for the study of adaptive clinal variation, but has also experienced a complicated history of range expansion and secondary contact following allopatric divergence, potentially obscuring the influence of contemporary evolutionary processes. Presumptively neutral genetic markers (microsatellites) demonstrated zones of secondary intergradation among coastal populations centred around northern New Jersey and the Chesapeake Bay region. Analysis of genetic variation indicated isolation by distance among some populations and provided supporting evidence that the Delaware Bay, but not the Chesapeake Bay, has acted as a barrier to dispersal among coastal populations. Bayesian estimates indicated large effective population sizes and low migration rates, and were in good agreement with empirically derived estimates of population and neighbourhood size from mark–recapture studies. These data indicate that populations are not in migration-drift equilibrium at a regional scale, and suggest that contributing factors include large population size combined with relatively low migration rates. These conditions should be considered when interpreting the evolutionary significance of the distribution of genetic variation among F. heteroclitus populations.