The English sole (Parophrys vetulus, family Pleuronectidae) is an abundant ground fish used as a sentinel of marine pollution in the inland marine waters of Washington and Oregon. We used 15 microsatellite loci to evaluate patterns of genetic variability within and among 17 collections of sole from coastal sites and within the southern Salish Sea. Over 470 alleles were identified in 15 loci, and high levels of genetic richness, as estimated by expected heterozygosity (H_E) and allele richness (A_R), were detected. Fish sampled from coastal areas at the northern end of our study area had the lowest values of genetic richness, in contrast to collections at the southern end. The level of differentiation among all collections was low: mean F_ST value was 0.0006. Nineteen comparisons had statistically significant F_ST values (mean F_ST of 0.0029) and involved comparisons involving three locations associated with urban areas (Port Gardner [Everett, WA], Sinclair Inlet [Bremerton, WA], and Commencement Bay [Tacoma, WA]). A statistically significant, but weak, isolation-by-distance pattern was seen in the five coastal collections over 1,100 km of coastline. In the Salish Sea collections, the pattern of differentiation was patchy and marked by several distinctive collections in close proximity to urban areas. The pattern of differentiation in the Salish Sea parallels differences seen in ecotoxicological characteristics of the English sole that inhabit separate urban embayments differing in pollution characteristics. Future work can focus on evaluating the stability and meaningfulness of this subtle variability in a time of local and global environmental changes.