The genetic diversity and differentiation of sea trout were studied in three river basins in Lithuania: Akmena-Dane, Bartuva, and Nemunas. A total of 282 individuals were genotyped at eight microsatellite loci. A similar level of genetic diversity was found in all of the populations studied: mean allelic richness ranged from 3.64 to 5.03, and average expected heterozygosity ranged from 0.588 to 0.721. Significant genetic divergence was observed among the different river basins as well as between populations within the drainages. All pairwise F (ST) values were highly significant, ranging from 0.027 to 0.197. The analysis of molecular variance showed rather weak hierarchical population structuring within the Nemunas basin, which may be explained by extensive gene flow among different river basins or, alternatively, reflect the influence of artificial breeding. Information on genetic diversity and differentiation of the Lithuanian sea trout populations will be useful for future management decisions.