Cotton gin trash (CGT) is a ubiquitous cotton-production-waste resource which can be used for ethanol production. In this research, seven combinations of three pretreatments; ultrasonication, liquid hot water and ligninolytic enzymes were evaluated on CGT to select the best pretreatments combination that increased the cellulose conversion and the ethanol yield in the saccharification and fermentation processes, respectively. The structural changes in the cellulose, hemicellulose and lignin from CGT were followed using FT-IR after each pretreatment. All the pretreatment combinations modified the CGT's structure and composition compared with the unpretreated CGT, and the majority of them improved release of sugars originally present in the CGT. The best results were achieved by the sequential combination of ultrasonication, hot water, and ligninolytic enzymes with an improvement of 10% in the ethanol yield and cellulose conversion compared to the other pretreatments. These results are a contribution to develop a feasible bioethanol production from CGT. (C) 2013 Elsevier Ltd. All rights reserved.