Different lipid extraction methods on fatty acids composition in cow milk. Comparative studies among 4 (four) different methods of total lipids extraction were carried out to evaluate the lipid extraction efficiency and fatty acids contents in cow milk. Total lipids extraction methods were Bligh e Dyer (BD), Folch et al. (FLS), Roese-Gottlieb (RG) and Gerber (GB). There were non-significant (p < 0.05) difference, in total lipids content among the extraction methods. The smallest concentrations of omega-3 (n-3) fatty acids and polyunsaturated fatty acids (PUFA) were observed on method GE, possibly due degradation of PUFA immersed in sulfuric acid used during analysis of total lipids. The highest concentrations of n-3 PUFA were observed by BD method, especially to omega-3/omega-6 ratio and alpha-linolenic acid (LNA, 18:3n-3), eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3), significant differences were observed among the methods. The results demonstrate that the different extractions influenced decisively on quantitative fatty acids composition and evaluations indicated the methods BD as better and GE as the worst to polyunsaturated fatty acids determination.