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The level of variation in lipids and their fatty acids was determined in the grains of 10 popular durum wheat cultivars commercially grown in Central and Southern Italy. Samples were harvested for two consecutive years to account for differences due to changes in climatic conditions. Total fat content was determined by means of the International Association of Cereal Science and Technology (ICC) Standard Method No. 136, whereas the fatty acid profile was determined by gas chromatography. Total lipid content ranged from 2.97% to 3.54% dry basis (d.b.) in the year 2010 and from 3.10% to 3.50% d.b. in the year 2011, and the average value was 3.22% d.b. considering both years together. Six main fatty acids were detected in all samples in order of decreasing amounts: linoleic (C18:2) > palmitic (C16:0) ≈ oleic (C18:1) > linolenic (C18:3) > stearic (C18:0) > palmitoleic (C16:1). Significant variations in the levels of single acids between two years were observed for three samples. These results will be very useful in the updating of food composition databases in general and will help authorities to set proper quality standards for wholegrain flours and products where the germ should be preserved, considering also the recent interest of industry and consumers for these kinds of products.