Dams have been built on waterways for centuries, acting as barriers to fish migration and possible impediments to gene flow. We tested whether the Munroe Falls low-head dam, constructed in 1817 on the middle Cuyahoga River in the northeast Ohio portion of the Lake Erie watershed, formed a barrier for fish migration. Subsequent to our sampling, the dam was removed in fall 2005. The present study characterized the population genetic composition of two species of darters (Family Percidae), Etheostoma blennioides (greenside darter) and Percina caprodes (logperch darter), upstream and downstream from the dam in comparison with an outlying population from the Grand River, Ohio using mitochondrial DNA control region and cytochrome b gene sequences. Results found a single genotype in the greenside darter samples that was shared upstream and downstream from the dam, and thus no evidence for effect of the dam could be discerned. In contrast, samples of logperch darter differed in genotypic frequencies above and below the dam, with unique alleles occurring below the dam. It thus is possible that the Munroe Falls low-head dam acted as a one-way barrier to gene flow for logperch. Further genetic studies should test for possible after-effects of the dam removal on these darter populations, and relate these data to variation along and among river systems, including other potential barriers to gene flow.