Abstract: The fatty acid composition in cottonseed of "Xinluzao 39" and "Xinluzao 50" methylated by three kinds of catalysts, KOH-methanol method, H₂SO₄-methanol and 14% BF₃-methanol methods, was determined by GC-MS. The quantitative standard curve was established by mixing 37 fatty acid methyl esters determinated by GC-MS, and the external standard method was used for quantitative determination. The results showed that there were 10 kinds of fatty acid composition in cottonseed oils, which could be detected on GC-MS analysis by the esterification of fatty acids with KOH-methanol. Only 6 kinds of fatty acid composition could be detected by H₂SO₄-methanol and 14% BF₃-methanol method. The results of GC-MS analysis after methyl esterification with KOH-methanol showed that the main fatty acids of cottonseed oil was linoleic acid, followed by palmitic acid and monounsaturated fatty acid of polyunsaturated fatty acids. The linoleic acid content per gram cottonseed was 147.29 mg and 163.2 mg in seed oil of Xinluzao 39 and Xinluzao 50, respectively, which accounted for 60.12% and 60.83% of its oil content. The oil also contained high level of palmitic acid, contents of which were 65.05 mg and 62.28 mg, accounting for 26.55% and 23.24% of their oil content respectively. The content of oleic acid was 24.79 mg and 22.19 mg, accounting for 10.12% and 8.28% of their oil content respectively. Among the three commonly used fatty acid methyl esterification methods, the KOH-methanol method was more convenient, rapid and had many kinds of tests. It was suitable for the methyl esterification of cottonseed fatty acids.