Hybridization with introduced or invasive species is a major threat and driver of population declines in native salmonids. The rainbow trout (Oncorhynchus mykiss, RBT) has been widely introduced globally and represents an important invasive species, often establishing entrenched naturalized populations. The cutthroat trout (Oncorhynchus clarkii, CT), a close congener, is particularly susceptible to competition and hybridization from RBT introductions which has led to range-wide population declines and loss of CT genetic variation. The Lahontan CT (O. c. henshawi, LCT) whose historic distribution included the Lake Tahoe basin, was extirpated by the 1940s due to overfishing and introduction of nonnative salmonids, including now naturalized RBT. Here, we characterize genetic variation of RBT in a subset of Lake Tahoe tributaries to assess potential homing of RBT to streams for spawning, thereby informing LCT reintroduction. Diploid reproductively viable RBT were stocked annually into Lake Tahoe from the late 1800s until the mid-2000s by California and Nevada fish and wildlife agencies, planting the same commonly raised hatchery strains over time. Since 2007, triploid RBT comprise the bulk of RBT planted. Despite extensive dispersal from stocking locations, our analyses revealed variation in population differentiation among tributaries, with individuals from spatially proximate streams clustering across multiple population genetic analyses. Although subtle, we detected evidence for genetic differentiation among tributaries from the southern, western, and northern regions, including surprising structure involving a single tributary. These results illustrate the extent of differentiation within and among streams and could inform possibilities for and implications of RBT removal and LCT reintroduction.