The addition of dams into a riverine system causes a wide range of changes (i.e., sedimentation, erosion, thermal) to the river as well as to the fish assemblages of that river. Although there have been many studies documenting the changes that occur to the fish assemblages in the impounded river, there have been fewer studies examining the effects of a reservoir on the fish inhabiting the tributaries upstream of the impoundment. One possible impact of a reservoir could be to act as a barrier to fish migration between streams. To determine if reservoirs restrict migration, the genetic diversity of two species of darter, the rainbow darter Etheostoma caeruleum Storer and the Highland Rim darter Etheostoma kantuckeense Ceas and Page, was determined from populations inhabiting the Barren River Lake drainage basin. Between ten and twenty-six individuals of each species were collected from each of 6 sites. Three streams were directly connected to Barren River Lake and three streams were directly connected to Barren River upstream of the reservoir. Allelic variation at 3 microsatellite loci was analyzed to determine the degree to which each population is isolated. If the reservoir is restricting gene flow between populations, the populations in streams adjacent to Barren River Lake would be predicted to have lower allele diversity and heterozygosity than those adjacent to the Barren River. Consistently high levels of allelic diversity (total number of alleles, N), observed heterozygosity (Ho), and effective number of alleles (Ae) across both reservoir and river study sites led to the rejection of the hypothesis that the reservoir is acting as a genetic barrier to darters. M-ratios differed between species, with Etheostoma caeruleum exhibiting consistently higher M--ratios than Etheostoma kantuckeense. The low M- seen in E. kantuckeense could be due to small sample sizes (largest sample for this species showed the highest M-), and could also be due to small natural populations. With the exception of Salt Lick Creek, high allelic diversity was observed at most sites for E. kantuckeense. A low M- coupled with high allelic diversity in most E. kantuckeense populations, may indicate that all of the study populations are recovering from a bottleneck event. These results indicate Etheostoma kantuckeense is sensitive to changes in the environment. When conservation agencies assess fish populations in South Central Kentucky, it is advantageous to know which species are currently at risk, which species are sensitive to environmental changes, and which species or populations are recovering from events that were detrimental to their genetic diversity.