Abstract: In order to search for fitting pretreatment methods in effective anaerobic fermentation of cotton stalks, effects of three pretreatments including high temperature, alkali treatment and microbial inoculation on biogas production by anaerobic fermentation of cotton stalks were studied. The pretreated cotton stalks were fermented in the biogas production experiments with total solid weight percentage of 8% under room temperature of (23±2)℃. The results showed that in cotton stalks pretreated with NaOH and microbial inoculation, the structure of lignocellulose was broken clearly, while high temperature affected the external structure indistinctively. All the three pretreatments effectively shortened the starting period of anaerobic fermentation, and improved the ability to biogas production of cotton stalks. Effects of the three pretreatments on biogas production followed the order that: alkali treatment > high temperature > microbial inoculation. Of all the conditions, the pretreatment of 4%(w/w) NaOH with 10 days treating period for cotton stalks (with 60% moisture) exhibited the best effect, which could gain the total gas production of 31 022.5 mL after 61 day fermentation. And in this fermentation, the daily average gas production achieved 508.57 mL/d, TS gas production and VS gas production reached 193.89 mL/g and 216.30 mL/g separately, which were much higher than those of the control and other pretreatments. This indicated that this kind of alkali treatment was the optimal, efficiently promoted biogas production in anaerobic fermentation of cotton stalks.