The absence of information on genetic variation and population structure of brook lampreys Lethenteron spp. in the eastern part of their distribution limits our understanding of the migration ecology and spatial population genetic structure of the species. We examined genetic variation within and among three aggregations of Lethenteron spp. larvae in the Yukon River drainage, Alaska, using microsatellite genotypes. A total of 120 larval lampreys were genotyped at eight microsatellite loci. Global FST was 0.053 (95% CI 0.021-0.086), while pairwise FST values ranged from 0.048-0.057). Model-based Bayesian clustering analyses with sample locality priors (LOCPRIOR) identified three distinct, but admixed, genetic clusters that corresponded with the three aggregations. Estimates of contemporary gene flow indicate substantial reciprocal migration among sites consistent with no or low-fidelity natal homing. These results are largely in agreement with previous reports of historic and contemporary gene flow among Lethenteron spp. in other parts of their geographic distribution.