Cotton stalks at four different particles sizes (<0.15 mm, 0.15-0.5 mm, 0.5-1.0 mm and 1.0-2.0 mm) were subjected to ionic liquid pretreatment via 1-ethyl-3-methylimidazolium acetate (EMIMAc) and 1-ethyl-3-methylimidazolium chloride (EMIMCl) followed by the enzymatic hydrolysis of pretreated biomass. The changes in crystalline structure, lignin content and digestibility of the pretreated samples were investigated in addition to the glucose yield obtained from cotton stalks. For EMIMAc pretreatment, the lowest glucose yield (57%) was obtained upon the pretreatment of cotton stalks having the smallest particle size prior to pretreatment (<0.15 mm) while cotton stalks with larger particle sizes gave higher glucose yields of which were at least 71%. On the contrary, EMIMCl pretreatment functioned more efficiently with cotton stalks having smaller particle sizes (<0.15 mm and 0.15-0.5 mm) where increasing the biomass particle size further caused a decrease in the glucose yield from approximately 49% to 33%, unlike the EMIMAc pretreatment. These differences in the glucose yields obtained for different ionic liquids suggest that the ideal particle size of biomass prior to pretreatment may change from one ionic liquid to another.