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Switchgrass, Panicum virgatum L., one of the three dominant grasses of the North American tall grass prairie, is a genetically and morphologically diverse species with an array of ploidy levels, or set of chromosomes, and ecotypes. The relationship between DNA content and ploidy level has been controversial. The objectives of this study were to provide clear photodocumentation of switchgrass chromosome numbers and to clarify the relationship between nuclear DNA content and chromosome number. Defining the relationship between ploidy level and nuclear DNA content will facilitate the use of molecular biology techniques, such as flow cytometry, in plant breeding and evolutionary biology. The switchgrass tetraploids examined, which contain 4 sets of chromosomes, had 36 chromosomes with a nuclear DNA content of 3.1 pg/nuclei, while octaploids (8 sets of chromosomes) had 72 chromosomes with 6.1 pg/nuclei. Tetraploid plants from lowland ecotypes had the same nuclear DNA content as tetraploid plants from upland ecotypes. Normal diploid chromosome pairing occurred at meiosis for all tetraploid and octaploid plants examined. Our results indicate that the lowland and upland ecotypes have the same basic genome, and that the octaploids most likely evolved from the tetraploids by a natural doubling of chromosomes, and did so long enough ago for meiosis to be stabilized. Further research is needed to explore the evolutionary origins of switchgrass.