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Periodic glaciation during the Quaternary period shaped the contemporary riverscape and distribution of freshwater fishes in the Mississippi River drainage of central North America. The rainbow darter (Etheostoma caeruleum) is a member of this ichthyofauna and has a disjunct distribution in glaciated and unglaciated environments west of the Mississippi River. The aim of this study was to utilize genome-wide SNP data to compare the population genomic structure of the rainbow darter in river networks with disparate glacial histories; the Volga River in the glaciated upper Mississippi River basin and the Meramec River in the unglaciated Ozark Plateau. Individuals were sampled from localities within each river system at distances dictated by the organismal life history and habitat preferences. Riverscape analyses were performed on three datasets: total combined localities of both rivers and one for each river independently. The results revealed a lasting influence of historic glaciation on the population genomic structure of rainbow darter populations. There was evidence of population expansion into the glaciated northern region following glacial retreat. The population genetic signature within the Volga River did not fit expectations of the stream hierarchy model, but revealed a pattern of repeated colonization and extirpation due to cyclic glaciation. The population within the unglaciated Meramec River adhered to the stream hierarchy model, with a directional order of genetic diversity based on the life history and habitat preferences of the species. These results demonstrate the necessity to consider the geologic and climatic history of a region as well as the life history of an organism when interpreting spatial genetic patterns.