Rainbow trout native to the McCloud River, California, USA (Oncorhynchus mykiss stonei) are thought to represent a relic, nonanadromous trout adapted to harsh, fragmented environments. These fish, commonly named McCloud River ‘redband’ trout, survive in their most primitive form in a small, spring-fed stream, Sheepheaven Creek, in the upper McCloud River drainage. Turn-of-the-century fisheries records document both coastal anadromous steelhead and freshwater resident trout within the McCloud River drainage. The phylogenetic position of the McCloud River redband trout within O. mykiss has been debated for over 50 years. Based on phenotypic evidence, these fish were first reported as ‘southern Sierra golden trout’ by Wales in 1939. Behnke (1970) considered them a relic subspecies of nonanadromous, fine-scaled trout. Allozyme and mitochondrial DNA evidence suggested a coastal lineage. In this study, we examined within- and among-basin genetic associations for Sheepheaven Creek redband trout using 11 microsatellite loci. Within-basin analyses supported unique genetic characteristics in Sheepheaven Creek’s trout in comparisons with other McCloud River rainbow trout. Microsatellite data supported significant independence between Sheepheaven Creek fish and hatchery rainbow trout. Inter-basin genetic distance analyses positioned Sheepheaven Creek fish with samples collected from Lassen Creek, a geographically proximate stream containing inland redband trout. California’s redband trout shared a close genetic association with Little Kern River golden trout (O.m. whitei) and isolated rainbow trout from Rio Santo Domingo, Baja, Mexico (O.m. nelsoni), suggesting a vicariant distribution of microsatellite diversity throughout the southern range of this species.