The population genetic structure of smallmouth bass, Micropterus dolomieu, in Lake Erie was investigated using two mitochondrial DNA sequences (control region and cytochrome b) and eight nuclear DNA microsatellite loci. The objective was to evaluate relative resolution of fine-scale versus broad-scale spawning population genetic structure across Lake Erie. Results showed that only cytochrome b sequences supported the divergence of populations grouped by basins, suggesting little correspondence to bathymetric features of Lake Erie. The majority of sampling sites were characterized by large within site variances, particularly with the microsatellite data, reducing the efficiency to delimit populations with the given sample sizes. Although conclusions from mtDNA and microsatellites were not corroborative, all data sets revealed some divergent sites across and within basins. The apparently weak genetic structuring of populations does not reflect the strong behavioral patterns of male nest-site fidelity and adult migration. Finer-scale structure among geographically proximate sites was detected primarily with microsatellite data. Several locations were consistently identified as most genetically divergent suggesting that they may serve as long-term attractor areas for spawning populations. Mitochondrial DNA data indicated a broader-scale pattern reflecting either colonization from at least two glacial refugia or different dispersal routes from a common refugium with subsequent genetic divergence through drift. Genetic variation of smallmouth bass in Lake Erie is likely a product of glacial history with behavioral and stochastic factors interacting at different spatial and temporal scales. A precautionary management approach would weigh both genetic and behavioral patterns and develop appropriate conservation strategies for a non-panmictic smallmouth bass population in Lake Erie.