River systems are vulnerable to natural and anthropogenic habitat fragmentation and will often harbor populations deviating markedly from simplified theoretical models. We investigated fine-scale population structure in the sedentary river fish Cottus gobio using microsatellites and compared migration estimates from three F-ST estimators, a coalescent maximum-likelihood method and Bayesian recent migration analyses. Source-sink structure was evident via asymmetry in migration and genetic diversity with smaller upstream locations emigration biased and larger downstream subpopulations immigration biased. Patterns of isolation by distance suggested that the system was largely, but not entirely, in migration-drift equilibrium, with headwater populations harboring a signal of past colonizations and in some cases also recent population bottlenecks. Up- vs. downstream asymmetry in population structure was partly attributable to the effects of flow direction, but was enhanced by weirs prohibiting compensatory upstream migration. Estimators of migration showed strong correspondence, at least in relative terms, especially if pairwise F-ST was used as an indirect index of relative gene flow rather than being translated to Nm. Since true parameter values are unknown in natural systems, comparisons among estimators are important, both to determine confidence in estimates of migration and to validate the performance of different methods.