Rainbow Trout Oncorhynchus mykiss in southwestern Alaska occupy coastal watersheds near the northern boundary of the species native range and support a world class wild trout sport fishery. Although low freshwater temperatures and a short growing season in this region may favor anadromy, these populations appear to exhibit a freshwater resident life history strategy. In this study we used genetic data to evaluate two hypotheses regarding the influence of the presumed migratory behavior of these Rainbow Trout on reproductive isolation among and within watersheds. The results were largely consistent with the predictions but there were exceptions. The data supported the hypothesis that the freshwater resident behavior precludes marine-mediated gene flow resulting in large genetic divergence and low admixture among watersheds. The estimate of F_CT (among-watershed differentiation, 0.350) was large and reflected over 96% of the variation among all sampled aggregations (F_ST = 0.363). However, evidence of admixed individuals in two adjacent watersheds and five first generation migrants among five watersheds suggests that the potential for coastal migration with gene flow exists in these populations. The data also supported the hypothesis that aggregations formed within watersheds during the spawning period (May-June) represent reproductively isolated populations. The pairwise estimates of F_ST and the G-test results revealed population structure in four of the six watersheds tested. However, not all aggregation pairs were found genetically distinct and there was notable variation in the pairwise F_ST estimates (0.000 – 0.067). In summary, the data reflected the predicted results for each hypothesis, but also revealed exceptions that, consistent with tagging studies, demonstrate the complexity of migratory behavior in southwestern Alaska Rainbow Trout. We discuss the implications of these results for fishery management and conservation.