The fractionation of butter oil to specific fractions is needed recently to multiply its uses and the requirement of its interchangeability. This article is concerning with the distribution of bioactive lipids in buffalo butter- oil and its fractions prepared by the dry fractionation technique. Materials and Methods: Thirty-two samples of fresh buffalo milk were collected, divided into 4 composite samples and then converted to butter oil. Each butter oil sample was fractionated to four fractions: solid fraction at 35°C (S35), solid fraction at 25°C (S25), solid fraction at 15°C (S15) and liquid fraction at 15°C (L15). Fatty acids profile was assayed included butyric acid (BA), short chain fatty acids (SCFAs), conjugated linoleic acid (CLA), odd & branched chain fatty acids (OBCFAs) and Trans fatty acids by using GC-MS apparatus. Results: Data revealed that L15 had a higher content of CLA as conjugated diene and triene fatty acids, while S25 contained higher content of total OBCFAs as compared to all other fractions. Conclusion: Dry fractionation lead to marked differences in the distribution of bioactive lipids in buffalo butter oil and its fractions. Each fraction could be used for specific purposes in food industries according to its melting properties and its healthy benefit