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  1. Identifying the evolutionary and ecological mechanisms that drive lineage diversification in the species-rich tropics is of broad interest to evolutionary biologists. Here, we use phylogeographic and demographic analyses of genomic scale RADseq data to assess the impact of a large geographic feature, the Amazon River, on lineage formation in a venomous pitviper, Bothrops atrox. We compared genetic differentiation in samples from four sites near Santarem, Brazil that spanned the Amazon and represented major habitat types. A species delimitation analysis identified each population as a distinct evolutionary lineage while a species tree analysis with populations as taxa revealed a phylogenetic tree consistent with dispersal across the Amazon from north to south. Phylogenetic analyses of mtDNA variation confirmed this pattern and suggest that all lineages originated during the mid- to late-Pleistocene. Historical demographic analyses support a population model of lineage formation through isolation between lineages with low ongoing migration between large populations and reject a model of differentiation through isolation by distance alone. Our results provide a rare example of a phylogeographic pattern demonstrating dispersal over evolutionary time scales across a large tropical river and suggest a role for the Amazon River as a driver of in-situ divergence by both impeding (butmore »not preventing) gene flow and through parapatric differentiation along an ecological gradient.« less