The genetic population structure of coastal cutthroat trout (Oncorhynchus clarki clarki) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies (O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial (FST = 0.121, RST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams (FSR = 0.054) and between regions (FRT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards’ chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.