In the last 100 years, the Bridle Shiner has declined over significant parts of its range. We used mitochondrial and nuclear microsatellite markers to investigate population structure of this species. Tissue samples were collected from populations in six drainages in PA, NJ, NY and CT. One predominant haplotype was observed in the Delaware, Housatonic, Passaic, and Raritan specimens. Specimens from the Hudson and St. Lawrence drainages had a separate unique haplotype. Microsatellite loci revealed low within-population genetic diversity and pairwise population comparisons of allelic divergence showed significant genetic differences among most drainages. Population structure analysis of microsatellite loci recover the same population clusters inferred using mtDNA. Within the Delaware drainage, there was significant differentiation among areas separated by 128 km. Several alternate scenarios of population divergence and population bottlenecks were investigated using approximate Bayesian computing. These supported a scenario with a bottleneck occurring in the ancestral population of Bridle Shiner followed by recent divergence of Northern and Southern drainages.