Switchgrass (Panicum virgatum L.) is a native North American prairie grass and is considered by many to be the most promising dedicated energy feedstock for the production of lignocellulosic ethanol. Switchgrass cultivars can be divided into two distinct classes or cytotypes. Upland varieties are typically octoploid and found in higher, more mesic sites while lowland varieties are typically tetraploid and found at lower, more hydric sites. While there has already been a substantial amount of research into optimizing different pretreatment methods for switchgrass, including Ammonia Fiber Expansion (AFEX), research up until this point has not focused on potential differences that could arise between the two cytotypes. For this experiment, AFEX pretreatment was performed at a number of different conditions on two varieties of switchgrass, Shawnee (upland) and Alamo (lowland), followed by enzymatic hydrolysis to determine sugar yields. The composition of the enzyme cocktail used during enzymatic hydrolysis was also varied to obtain the optimal sugar yields.