Abstract    BACKGROUND: Industrial method of butter production involves direct separation of butter fat from the milk, however in traditional-making process of butter and oil in rural regions of Kermanshah province in west of Iran, the milk is converted to the yoghourt by the fermentation and following a few days maintenance of the yoghourt at room temperature, the butter fat is separated from the resulted yoghourt using several hours vigorous shaking. After melting of the butter and separation of its aqueous contents, traditional oil is prepared. A large body of evidence indicate that the type of fat has greater importance than total amounts of consumed fats with respect to risk of coronary artery disease (CAD), hence this study aimed to evaluate trend of fatty acids changes and cholesterol content from milk to oil, during the traditional method.    METHODS: Samples of milk, yoghourt, butter and oil prepared from the same bulk of milk were collected from different rural regions of Kermanshah province. To compare the traditional and industrial methods, samples of the industrially–prepared butter were purchased commercially. The total lipids of samples were extracted and subjected to fatty acids analysis by high performance liquid chromatography (HPLC).    RESULTS: We found that significant reduction is taking place in the cholesterol and long chain fatty acids contents of butter and oil during the traditional method, while short- and medium- chain fatty acids are significantly increased.  The fatty acid composition of industrially-made butter in our study however, was the same as that of the milk samples.      CONCLUSION: Compared with the industrial method, the fatty acid composition of butter in the traditional method has better nutritional value. Some aspects of this process may be recommended for improvement of the commercial methods of butter production.      Keywords: Fatty acid, Cholesterol, Butter, Oil, Nutrition.