Feedstock quality mainly depends upon the biomass composition and bioenergy conversion system being used. Higher cellulose and hemicellulose concentrations are desirable for biochemical conversion, whereas higher lignin is favored for thermochemical conversion. The efficiency of these conversion systems is influenced by the presence of high nitrogen and ash concentrations. Switchgrass (Panicum virgatumL.) varieties are classified into two ecotypes based on their habitat preferences, i.e., upland and lowland. The objectives of this study were to quantify the chemical composition of switchgrass varieties as influenced by harvest management, and to determine if ecotypic differences exist among them. A field study was conducted near Ames, IA during 2012 and 2013. Upland (‘Cave-in-Rock’, ‘Trailblazer’ and ‘Blackwell’) and lowland switchgrass varieties (‘Kanlow’ and ‘Alamo’) were grown in a randomized block design with six replications. Six biomass harvests were collected at approximately 2-week intervals each year. In both years, delaying harvest increased cellulose, hemicellulose and lignin concentrations while decreasing nitrogen and ash concentrations in all varieties. On average, Kanlow had the highest cellulose and hemicellulose concentration (354 and 321 g kg−1 DM respectively), and Cave-in-Rock had the highest lignin concentration (33 g kg−1 DM). The lowest nitrogen and ash concentrations were observed in Kanlow (14 and 95 g kg−1 DM respectively). In general, our results indicate that delaying harvest until fall improves feedstock quality, and ecotypic differences do exist between varieties for important feedstock quality traits. These findings also demonstrate potential for developing improved switchgrass cultivars as bioenergy feedstock by intermating lowland and upland ecotypes.