Abstract: The cottonseed oil was extracted by Soxhlet extraction. The fatty acids in the cottonseed oil were esterified by two methods and analyzed by gas chromatography mass spectrometry (GC/MS). The components and their contents of the fatty acids were compared with each other. The results showed that 11 fatty acids were identified by esterification method 1 and 7 fatty acids were identified by esterification method 2, the contents of the identified fatty acids make up 96.16% and 98.32% of the cottonseed oil respectively. A comparison of the major fatty acids was made: the major fatty acids were hexadecanoic acid (27.26%), 9,12 octadecadienoic acid (40.24%) and 9 octadecenoic acid (14.27%) by esterification method 1; the major fatty acids were hexadecanoic acid (27.12%), 9,12 octadecadienoic acid (48.00%) and 9 octadecenoic acid (15.51%) by esterification method 2.