Catchment population structure and divergence patterns of the rainbow darter Etheostoma caeruleum (Percidae: Teleostei), an eastern North American benthic fish, are tested using a landscape genetics approach. Allelic variation at eight nuclear DNA microsatellite loci and two mitochondrial DNA regions [cytochrome (cyt) b gene and control region; 2056 aligned base pairs (bp)] is analysed from 89 individuals and six sites in the Lake Erie catchment (Blanchard, Chagrin, Cuyahoga and Grand Rivers) v. the Ohio River catchment (Big Darby Creek and Little Miami River). Genetic and geographic patterning is assessed using phylogenetic trees, pair-wise F(ST) analogues, AMOVA partitioning, Mantel regression, Bayesian assignment, 3D factorial correspondence and barrier analyses. Results identify 34 cyt b haplotypes, 22 control region haplotypes and 137 microsatellite alleles whose distributions demonstrate marked genetic divergence between populations from the Lake Erie and Ohio River catchments. Etheostoma caeruleum populations in the Lake Erie and Ohio River catchments diverged c. 1.6 mya during the Pleistocene glaciations. Greater genetic separations characterize the Ohio River populations, reflecting their older habitat age and less recent connectivity. Divergence levels within the Lake Erie catchment denote more recent post-glacial origins. Notably, the western Lake Erie Blanchard River population markedly differs from the three central basin tributary samples, which are each genetically distinguishable using microsatellites. Overall relationships among the Lake Erie sites refute a genetic isolation by geographic distance hypothesis. Etheostoma caeruleum populations thus exchange few genes and have low migration among tributaries and catchments.