Cotton stalks (CS) are considered a good candidate for fuel-ethanol production due to its abundance and high carbohydrate content, but the direct conversion without pretreatment always results in extremely low yields due to the recalcitrant nature of lignocelluloses. The present study was undertaken to investigate the effect of various chemical and physicochemical pretreatment methods, i.e., alkali, microwave-assisted acid, organosolv, hydrothermal treatment, and sequentially organosolv and hydrothermal pretreatment, on chemical composition of CS and subsequent ethanol production applying pre-hydrolysis and simultaneous saccharification and fermentation (PSSF) at high solid loading. The best results in terms of ethanol production were achieved by the sequential combination of organosolv and hydrothermal pretreatment (32.3 g/L, using 15% w/v substrate concentration and 6 h pre-hydrolysis) with an improvement of 32% to 50% in ethanol production compared to the other pretreatments. Extending pre-hydrolysis time to 14 h and increasing substrate concentration to 20% w/v, ethanol production reached 47.0 g/L (corresponding to an ethanol yield of 52%) after 30 h of fermentation.