Agronomic and physiological evaluation of potential breeding lines of switchgrass (Panicum virginatum L.) is critical in identifying new genotypes with economically viable traits of high biomass and quality. The objective of this study was to evaluate genotypic variation and genotype x environment (G x E) interaction for agronomic, physiological and biofuel traits among 19 new switchgrass populations and two check cultivars. Simultaneous experiments were established both in greenhouse and multi-locations (Chickasha, Lane, Stillwater, Tipton and Woodward) in Oklahoma. Results from greenhouse study showed genotypic variation for different morphological and physiological traits. Higher stem elongation rates were observed in Southern Lowland (SL) and Northern Southern Lowland (NSL) genotypes whereas Southern Northern Upland (SNU) Early and Late Maturing genotypes showed lower stem elongation rates. Lowland populations showed high stem elongation and tiller elongation rates relative to the upland populations, but high tillering rate was observed in Upland populations. Genotype SL 93 C₂-1 stem elongation rate and genotype SL 93 C₂-2  photosynthetic capacity were 12% and 7% respectively greater over the existing high yielding check cultivar Alamo. Switchgrass populations performed differently across the locations, for plant height, tiller number, stand density, and biomass, in the multi-location trial. The preliminary results from an establishment year were evident for the G x E interaction among switchgrass populations. Multi-year evaluations could provide better understanding of G x E interaction and will help identify switchgrass lines adapted to a broad range of environments.