Steelhead trout, Oncorhynchus mykiss (the anadromous form of rainbow trout), are declining over much of their range around the Pacific rim. We nondestructively collected tissue samples from 494 adult steelhead from eight tributaries and two mainstem river sites within three watersheds in northern British Columbia, Canada. We scored allele size for six highly polymorphic microsatellite DNA loci and provide primer sequences and polymerase chain reaction conditions for five of these loci for the first time. The populations were significantly genetically differentiated (theta = 0.039; 95% confidence = 0.030-0.053). AMOVA showed that most of the genetic variation was at the individual level (95.6%), although significant genetic variation existed at the tributary level (3.09%) and watershed level (1.31%). The calculated unbiased genetic distances were positively correlated with geographical distance within watersheds (P < 0.01; r(2)=0.35) indicating probable genetic equilibrium. Tributary populations in two of the watersheds were not as genetically divergent as would be expected given their large geographical separation. Cross-headwater transfers of fish within relatively recent history are the most likely explanation of this anomaly. Seven of the eight tributary populations fit a regression line of mean heterozygosity vs. rearing habitat area. The one anomalous population had a much lower heterozygosity than expected based on the linear regression, and may thus be the population of greatest conservation concern.