Switchgrass (Panicum virgatum L.) is an important crop for bioenergy feedstock development. Switchgrass has two main ecotypes: the lowland ecotype being exclusively tetraploid (2n = 4x = 36) and the upland ecotype being mainly tetraploid and octaploid (2n = 8x = 72). Because there is a significant difference in ploidy, morphology, growth pattern, and zone of adaptation between and within the upland and lowland ecotypes, it is important to discriminate switchgrass plants belonging to different genetic pools. We used 55 simple sequence repeats (SSR) loci and six chloroplast sequences to identify patterns of variation between and within 18 switchgrass cultivars representing seven lowland and 11 upland cultivars from different geographic regions and of varying ploidy levels. We report consistent discrimination of switchgrass cultivars into ecotype membership and demonstrate unambiguous molecular differentiation among switchgrass ploidy levels using genetic markers. Also, SSR and chloroplast markers identified genetic pools related to the geographic origin of the 18 cultivars with respect to ecotype, ploidy, and geographical, and cultivar sources. SSR loci were highly informative for cultivar fingerprinting and to classify plants of unknown origin. This classification system is the first step toward developing switchgrass complementary gene pools that can be expected to provide a significant heterotic increase in biomass yield.