Cotton stalk can be used as a reinforcement material for biocomposite production and it can be evaluated for biofuel production. The aim of this research is to investigate the effect of different chemical pretreatments on sugar production and fiber production from cotton stalks. For this purpose, thermal assisted alkaline pretreatment, microwave pretreatment with sulfuric acid, and hydrochloric acid pretreatments were performed by varying temperature, reaction time, and concentration. The results were compared according to total sugar amount and cellulose content. The highest amount of total sugar of thermal assisted alkaline pretreatment was 9.98 g/L, microwave pretreatment was 84.80 g/L, and hydrochloric acid pretreatment was 36.58 g/L. Also, the highest amount of cellulose of thermal assisted alkaline, microwave, and hydrochloric acid pretreatment were 64.12%, 62.54%, and 64.12%, respectively. Thus, while microwave pretreatment was the most effective method for total sugar, both alkaline and hydrochloric acid pretreatment had the same highest result for cellulose.