Technical Abstract: Big bluestem (Andropogon gerardii Vitman) is a native grass of the North American tallgrass prairie, used as forage crop and under development as a biofuel feedstock. In the current study, genetic diversity was measured both among and within three groups of big bluestem populations: natural populations collected in Wisconsin, natural populations from the northeast USA, and cultivars or accessions available through various gene bank organizations. A total of 417 amplified fragment length polymorphism (AFLP) DNA markers and six chloroplast DNA (cpDNA) regions were used for principle component analysis, analysis of molecular variance (AMOVA), Mantel tests, and haplotype analysis. Results indicate that the germplasm groups represent three distinct yet overlapping genetic pools. Partitioning of genetic variance for each of the three groups revealed a significant amount of variance among ecoregions and hardiness zones, and among populations within ecoregions and hardiness zones. Although significant, the percentage of explained variance was generally small. One exception was among populations for samples originating in the Northeast (20% of the variance). Additionally geographic distance and climatic variables were found to be correlated with genetic distance for samples originating in the Northeast. We conclude that unique germplasm of big bluestem is present outside of Great Plains, providing opportunities to increase genetic variance within current breeding germplasm and introduce new alleles affecting traits of interest.