Microsatellite genetic diversity found in San Francisquito Creek rainbow trout support a close genetic relationship with rainbow trout (Oncorhynchus mykiss) from another tributary of San Francisco Bay, Alameda Creek, and coastal trout found in Lagunitas Creek, Marin County, California. Fish collected for this study from San Francisquito Creek showed a closer genetic relationship to fish from the north-central California steelhead ESU than for any other listed group of O. mykiss. No significant genotypic or allelic frequency associations could be drawn between San Francisquito Creek trout and fish collected from the four primary rainbow trout hatchery strains in use in California, i.e. Whitney, Mount Shasta, Coleman, and Hot Creek hatchery fish. Indeed, genetic distance analyses (δµ²) supported separation between San Francisquito Creek trout and all hatchery trout with 68% bootstrap values in 1000 replicate neighbor-joining trees. Not surprisingly, California hatchery rainbow trout showed their closest evolutionary relationships with contemporary stocks derived from the Sacramento River. Wild collections of rainbow trout from the Sacramento-San Joaquin basin in the Central Valley were also clearly separable from San Francisquito Creek fish supporting separate, independent ESUs for two groups of O. mykiss (one coastal and one Central Valley) with potentially overlapping life histories in San Francisco Bay. These data support the implementation of management and conservation programs for rainbow trout in the San Francisquito Creek drainage as part of the central California coastal steelhead ESU.