Oats are considered the cereal of the future due to their unique lipid composition compared to other cereals. Since the production origin and genotype can affect the fatty acid composition of oat lipids, this study evaluated the lipid content and fatty acid composition of three Avena sativa oat samples collected from three different sub-districts of the Awi zone, one of the main oat producing areas of the Amhara region. The oat samples were collected using a purposive sampling technique during the 2022 crop season from three sub-districts of Ankesha District in the Awi Zone: Den Zuria, Dangula, and Urana. The Folch method was used to extract the lipids, and the acid-catalyzed derivatization method was employed to convert the fatty acids into fatty acid methyl esters (FAME). Gas chromatography-mass spectrometry (GC-MS) was utilized to determine the fatty acids. The results revealed that all oat samples contained a crude fat content of 4.23%, 5.64%, and 4.95% by weight, respectively, in samples collected from Den Zuria, Dangula, and Urana. In addition, in these samples, it was noted that there were a total of thirteen fatty acids, seven of which were unsaturated and six of which were saturated. The most common fatty acid concentrations in the three sub-district samples from Ankesha District were palmitic, linolenic, oleic, stearic, rumenic, gadoleic, and arachidic acids. Palmitic acid was the most abundant fatty acid in the oats, accounting for 43.4-49.6% of the total fatty acid content. The second and third most abundant fatty acids in the oat samples were linoleic and oleic acids, constituting 24.7-28.5% and 8.86-11.6%, respectively. Although the relative abundances of each fatty acid in the three sampling sites are insignificant, nutrient-wide variations are shown in the fatty acid profiles in Den Zuria, Urana, and Dangula oat varieties. The major one was palmitic acid, with as high as 363mg/100g in Dangula, linoleic acid ranged from 129 to 233 mg/100g, and linolenic acid ranged from 32.1 to 56.9 mg/100g. This may indicate that oat is a useful source of nutritionally important fatty acids and hence its consumption is beneficial to cardiovascular health. Further research would be done to find out if these fatty acids have health implications when included in the diet.This study noted that there is a slight variation in crude oil content and minor fatty acids across the sampling districts, but the seeds are richer in fatty acid and crude fat content compared to the common cereals such as barley and wheat reported elsewhere. Thus, oats could be a good source of crude fat for consumers. Keywords: oats, fatty acids, gas chromatography-mass spectrometry (GC-MS), Crude fat