Switchgrass (Panicum virgatum L.) is a native North American prairie grass being developed for bioenergy production in the central and eastern USA. The objective of this study was to identify the impacts of harvest time and switchgrass cultivar had on sugar release variables determined through enzymatic hydrolysis. Previously, we reported that delaying harvest of switchgrass until after frost and until after winter resulted in decreased yields of switchgrass but it reduced the amount of ash and nutrients in the biomass. The current study used near-infrared reflectance spectroscopy (NIRS) to broaden an existing set of calibration equations designed to predict composition and sugar release variables of switchgrass. These updated calibrations were then applied to the full set of samples from a multi-year and multi-location switchgrass harvest-management study. Composition and processor sugar yields were significantly affected by location, year, cultivar, and harvest time, of which the time of harvest was the most important. Delaying the time of harvest until after frost or post-winter increased the concentration of structural carbohydrates from 500 to over 570 g kg−1 in the biomass and lignin content from 160 to over 200 g kg−1. Conversely, delaying harvest time lowered the amounts of ash and soluble sugars. The later harvest times also yielded more sugars following processing with yields increasing over 20% from the first harvest. Increased sugar yields are attributable to both increased concentration of sugars in the biomass upon harvest and reduced biomass recalcitrance. Based upon processed sugar yields, it is estimated that a biorefinery producing 76 million liters of ethanol per year would require 229–373 km2 of land cultivated with switchgrass.