Genetic variation at 11 microsatellite loci described population genetic structure for Oncorhynchus mykiss in the Central Valley, California. Spatial and temporal variation was examined as well as relationships between hatchery and putative natural spawning anadromous stocks. Genetic diversity was analyzed at two distinct spatial scales: fine-scale within drainage for five populations on Clear Creek; between and among drainage diversity for 23 populations. Significant regional spatial structure was apparent, both within Clear Creek and among rainbow trout populations throughout the Central Valley. Significant differences in allelic frequencies were found among most river or drainage systems. Less than 1% of the molecular variance could be attributed to differences found between drainages. Hatchery populations were shown to carry similar genetic diversity to geographically proximate wild populations. Central Valley M = 0.626 (below the M < 0.68 threshold) supported recent population reductions within the Central Valley. However, average estimated effective population size was relatively high (Ne = 5066). Significant allelic differences were found in rainbow trout collected above and below impassable dams on the American, Yuba, Stanislaus and Tuolumne rivers. Rainbow trout sampled in Spring Creek were extremely bottlenecked with allelic variation at only two loci and an estimated effective population size of 62, suggesting some local freshwater O. mykiss stocks may be declining rapidly. These data support significant genetic population structure for steelhead and rainbow trout populations within the Central Valley across multiple scales. Careful consideration of this genetic diversity and its distribution across the landscape should be part of future conservation and restoration efforts.