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Quantifying spatial genetic structure can reveal the relative influences of contemporary and historic factors underlying localized and regional patterns of genetic diversity and gene flow - important considerations for the development of effective conservation efforts. Using 10 polymorphic microsatellite loci, we characterize genetic variation among populations across the range of the Eastern Sand Darter (Ammocrypta pellucida), a small riverine percid that is highly dependent on sandy substrate microhabitats. We tested for fine scale, regional, and historic patterns of genetic structure. As expected, significant differentiation was detected among rivers within drainages and among drainages. At finer scales, an unexpected lack of within-river genetic structure among fragmented sandy microhabitats suggests that stratified dispersal resulting from unstable sand bar habitat degradation (natural and anthropogenic) may preclude substantial genetic differentiation within rivers. Among-drainage genetic structure indicates that postglacial (14 kya) drainage connectivity continues to influence contemporary genetic structure among Eastern Sand Darter populations in southern Ontario. These results provide an unexpected contrast to other benthic riverine fish in the Great Lakes drainage and suggest that habitat-specific fishes, such as the Eastern Sand Darter, can evolve dispersal strategies that overcome fragmented and temporally unstable habitats. [ABSTRACT FROM AUTHOR]