Seven microsatellite DNA loci were optimized to assess genetic differentiation in coastal steelhead (Oncorhynchus mykiss irideus) sampling groups from the lower Klamath River (California, USA). Genetic relationships among three winter-run and two summer-run groups were investigated. The different groups displayed high levels of allelic variation. Pairwise FST comparisons and Nei's genetic distance supported low, yet significant, genetic differentiation between summer and winter run-timings similar to other studies of temporal variation in salmonids. Analysis of molecular variance showed that most of the genetic variation was at the individual level (97.9%), although significant genetic variation existed between timing of runs (2.59%). Additionally, at least one locus in each group was out of Hardy–Weinberg equilibrium due to a deficiency in heterozygotes, and significant FIS values were observed in three temporal collections. Together, these results suggest stock admixture, caused by multiple populations of origin in each sampling group, better known as the Wahlund Effect. These observations are preliminary evidence for isolation by time between Klamath River steelhead runs during distinct periods.