Switchgrass (Panicum virgatum L.), is a warm season C₄ perennial grass, native to North American tall grass prairies that is widely grown for summer grazing and soil conservation. The high biomass production potential of switchgrass with low inputs makes it an excellent choice as a sustainable bio-energy crop. The objective of this study was to determine the genetic diversity of within and among 32 switchgrass populations including Plant Introductions (PIs) and four cultivars. Six plants from each population (192 genotypes) were characterized with simple sequence repeat (SSR) markers developed from conserved grass (CG) and switchgrass expressed sequence tags (ESTs). The pair-wise genetic distance estimates based on SSR data revealed dissimilarity coefficients ranging from 0.32 to 0.80. Cluster analysis grouped the 192 genotypes from 32 populations into four major groups. The uplands and the lowlands were concentrated in two distinct groups; a third group consists of germplasms collected from the eastern states of USA. Genotypes from an Arkansas collection were found to be very distinct. The partitioning of variance components based on the analysis of molecular variance (AMOVA) revealed between population variability (20%), but the variability within population was significantly higher (80%) than between the populations. This study will be helpful in identifying diverse collections that could benefit the genetic improvement of switchgrass.