Abstract Hatchery rainbow trout Oncorhynchus mykiss have been stocked in the Metolius River in central Oregon since 1938, and legal-sized (≥160 g) yearling trout were stocked annually from 1947 until 1995. In 1996, management objectives shifted to emphasize wild trout, and hatchery stocking ceased. We examined allozyme and mitochondrial DNA (mtDNA) variation among three naturally occurring populations of rainbow trout in the Metolius River to investigate possible hybridization with hatchery-produced rainbow trout. We also examined two commonly used hatchery strains of rainbow trout, one of which has supplied nearly all of the catchable hatchery trout in the Metolius. Both allozyme and mtDNA data showed the two hatchery samples to have genetic characteristics typical of hatchery populations derived from coastal rainbow trout O. mykiss irideus. Rainbow trout sampled from the lower Metolius River, approximately 30 km downstream of the headwaters, had allozyme and mtDNA characteristics typical of interior rai...