Determining fine‐scale genetic diversity and structure is critical for the conservation and management of populations, especially those under heavy anthropogenic influence. We analyzed 446 individuals at nine microsatellite loci to determine the local population structure of naturally produced steelhead Oncorhynchus mykiss and genetic differentiation from introduced hatchery strain steelhead in the Klickitat River of the Pacific Northwest. We detected significant genetic structure among steelhead in various tributaries to the Klickitat River; the most divergent population was located above a waterfall that acts as a partial upstream migration barrier (average pairwise FST = 0.13; P < 0.0001). Analysis of mixtures indicated an estimate of six to seven genetically distinct populations of naturally reproducing steelhead in this river system. The hatchery strain appears to remain genetically distinguishable from native stocks (average pairwise FST of 0.078 with P < 0.0001), as only 4.0% of naturally produced steelhead had their most likely assignment to the hatchery strain. These results indicate that the genetic integrity and variation of native Klickitat River steelhead have been maintained despite repeated hatchery introductions and that the potential is high for restoring this threatened population. Further, this study suggests that hierarchical analyses of mixtures to identify distinct populations in a watershed are a valuable method for directing management of reproductively isolated populations.

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