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Switchgrass, Panicum virgatum L., is a native, cross-pollinated, morphologically diverse species with an array of ploidy levels and ecotypes. Switchgrass is found throughout most of the USA and Canada, primarily east of the Rocky Mountains and south of Hudson Bay. The objective of this research was to determine if chloroplast DNA (cpDNA) restriction fragment length polymorphisms (RFLPs) occur among switchgrass cultivars and experimental strains that differ in ploidy level or ecotype classification. Eighteen switchgrass cultivars or experimental strains representative of reported ecotypes, ploidy levels, and the geographical range of switchgrass were surveyed for cpDNA polymorphisms by means of four restriction endonucleases and 20 sorghum cpDNA probes. One polymorphism was detected which was associated with the lowland-upland ecotype classification. The lowland cultivars contained a restriction site change that was not present in the upland cultivars. The two cytotypes discovered have been designated as the U (upland) or L (lowland) cytotype. The lowland cultivars had 3 pg DNA/nuclei as measured by flow cytometry while the upland types had either 3 or 6 pg DNA/nuclei. There were no cpDNA polymorphisms among the upland cultivars regardless of ploidy level as measured by DNA content. These results demonstrate that cpDNA differs among switchgrasses and that this variation is associated with ecotype variation but not with nuclear DNA content.