Microsatellite DNA variation was examined in rainbow trout ( Oncorhynchus mykiss ) populations from throughout British Columbia, Canada, to address the roles of historical isolation, postglacial dispersal, and contemporary geomorphology in structuring genetic variation and differentiation. We detected signatures of historical isolation and postglacial recolonization in the form of “interior” and “coastal” population groupings, a decline in genetic variation as distance increased from putative glacial refugia, and different extents of isolation-by-distance in different regions. Rainbow trout populations were structured genetically into major regions and into smaller watersheds and then into drainages. Within drainages, high levels of dispersal and gene flow were inferred between geographically proximate and contiguous lakes. Elevation, stream branching points (nodes), fluvial distance, migration barriers, and stream and lake order influenced genetic diversity within, and differentiation among, populations. Habitat characteristics, particularly lake surface area and perimeter, were poor predictors of genetic variation. Although founder events and postglacial dispersal influenced broadscale patterns of genetic diversity in rainbow trout, our results suggest that contemporary factors can strongly modulate historical patterns.