Switchgrass (Panicum virgatum L.), a warm season grass native to North America, is being developed as a biofuel crop. Plant breeding can improve biofuel characteristics further, particularly if the genetic diversity of germplasm resources is clearly understood. The objective of this study is to examine the population structure and relatedness within and among 36 switchgrass populations mainly derived from the southern United States coupled with the examination of phenotypic diversity and its relation with genetic subpopulation structure. A total of 464 genotypes were genotyped using thirty-five SSR markers and a chloroplast specific marker to assess the genetic structure of the germplasm. Population structure analysis showed the expected clear separation into two subpopulation groups representing upland and lowland ecotypes. A further substructuring of the population was evident within both upland and lowland groups, based largely, but not entirely, on geographical location of origin. The chloroplast specific marker similarly differentiate upland and lowland ecotypes with some exceptions. The preliminary results from the phenotypic analyses of several traits showed high diversity for each trait. Collectively, nuclear DNA and chloroplast markers and phenotypic data of key traits helped to classify switchgrass accessions into sensible subgroups. This information is critical to the development of improved cultivars and to the successful application of association mapping in switchgrass.