Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods.