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Understanding genetic diversity across large spatial scales helps to reveal patterns of population structure. Mitochondrial DNA sequences and microsatellite loci were used to analyze the phylogeography of a common unionid species (Lasmigona costata) from the Laurentian Great Lakes and historically connected river drainages. Phylogeographic patterns were assessed to determine colonization routes into the Great Lakes following glacial recession. A suite of seven microsatellite loci were genotyped and a fragment of the mitochondrial gene COI was sequenced. Multiple analyses using microsatellite allele frequencies suggest at least two distinct genetic populations for L. costata. A total of seven hypothesized post-glacial dispersal scenarios were compared using isolation by distance to test the various dispersal models. Evidence was strongest for two post-glacial dispersal routes into the Great Lakes: one utilizing a connection between the Wabash and Maumee River watersheds, and one utilizing a connection between the Wisconsin River and Green Bay watersheds. A highly differentiated and monophyletic population of L. costata was identified in the Ozark Highlands, which may constitute a unique taxonomic entity. [ABSTRACT FROM AUTHOR]