AbstractWe used 13 microsatellite loci to examine population structure in rainbow trout Oncorhynchus mykiss collected from 20 tributaries and 3 main stems in the greater Spokane River drainage. Populations displayed some excess homozygosity and linkage disequilibrium, which was more pronounced in upper tributary collections and probably the result of small effective population sizes or structuring within tributaries. In general, population structure followed geographic structure; collections from creeks within sub‐drainages were the most closely related, and collections from different tributaries were genetically distinct. Comparisons with cutthroat trout O. clarkii indicated little to no introgression. Comparisons with steelhead (anadromous rainbow trout), coastal rainbow trout O. mykiss irideus, and inland rainbow trout from hatcheries suggested introgression by hatchery fish into some wild populations. Introgression was suspected in populations from stocked tributaries and tributaries that lacked barriers to escaped hatchery fish. Populations from tributaries above barriers that had not been stocked were genetically distinct from hatchery fish and appeared to be native inland redband trout O. mykiss gairdneri.