Restoration of access to lost habitat for threatened and endangered fishes above currently impassable dams represents a major undertaking. Biological monitoring is critical to understand the dynamics and success of anadromous recolonization as, in the case of Oncorhynchus mykiss, anadromous steelhead populations are reconnected with their conspecific resident rainbow trout counterparts. We evaluate three river systems in the Lower Columbia River basin: the White Salmon, Sandy, and Lewis rivers that are in the process of removing and/or providing passage around existing human-made barriers in O. mykiss riverine habitat. In these instances, now isolated resident rainbow trout populations will be exposed to competition and/or genetic introgression with steelhead and vice versa. Our genetic analyses of 2,158 fish using 13 DNA microsatellite (mSAT) loci indicated that within each basin anadromous O. mykiss were genetically distinct from and significantly more diverse than their resident above-dam trout counterparts. Above long-standing natural impassable barriers, each of these watersheds also harbors unique rainbow trout gene pools with reduced levels of genetic diversity. Despite frequent releases of non-native steelhead and rainbow trout in each river, hatchery releases do not appear to have had a significant genetic effect on the population structure of O. mykiss in any of these watersheds. Simulation results suggest there is a high likelihood of identifying anadromous x resident individuals in the Lewis and White Salmon rivers, and slightly less so in the Sandy River. These genetic data are a prerequisite for informed monitoring, managing, and conserving the different life history forms during upstream recolonization when sympatry of life history forms of O. mykiss is restored.