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Tobacco (Nicotiana tabacum L.) is an important industrial crop and its seeds contain significant amounts of oil. The extraction of oil components using solvent at high pressure, or supercritical fluid (SCF), has received much attention. In the present study, statistical analyses showed that the average extraction yield of seed oil of five tobacco varieties using SFE was 9.33%, which was higher than Sonication (7.75%) and DGF (Deutsche Gesellschaft f_r Fettwissenschaft) standard method B-I5(87) (8.48%), but lower than Soxhlet (13.72%). Also, fatty acids profile of each extracted oil was determined by gas chromatography. Various saturated and unsaturated fatty acids such as lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and eicosanoic (C20:0) acids were observed in the extracted oils.