Significant genetic diversity was revealed for Oregon and California steelhead populations using 10 microsatellite loci. Fisher’s exact tests for population independence supported strong genetic substructure in sample populations at the southern extent of this species range. Population genetic structure depicted with these loci strongly supports biogeographic structure in coastal steelhead throughout California and reveals significant separation of Oregon and California populations into distinct evolutionary units. Two tests of isolation-by-distance support clear biogeographic structure in California steelhead populations and southern steelhead hatchery stocks retain genetic similarity to their original parental stocks in the Sacramento River. Differentiation of Sacramento River and San Joaquin River populations was , however, not possible using these 10 loci. AMOVA analysis of the genetic diversity found at these ten loci showed that 88.8% of the diversity was within populations. Only 8.5% of the allelic diversity was found among populations, and 2.7% was found among large geographic regions. Altogether the results indicated that these microsatellite loci are valuable tools for addressing fine scale population genetic questions at the basin level, for differentiation of wild stocks, or looking at hatchery vs. wild interactions. More loci are needed, however, to answer questions of life history differences between rainbow trout at the southern most reaches of their range.