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To select and introduce the best rapeseed genotypes under withholding irrigation, two field experiments were carried out in seasons 2015–2016 and 2016–2017 in Iran. Factorial arrangement of treatments was set up as RCBD with three replications. The experiment factors were two irrigation treatments (normal irrigation during the growing season and withholding irrigation from silique setting stage until the end of the growing season) and 17 genotypes of rapeseed. Grain yield, oil content and fatty acid contents were measured. The results revealed that oleic acid (62.15%) was the highest followed by linoleic (19.28%), linolenic (5.65%), palmitic (5.24%) and stearic acids (2.44%). ODR (oleic desaturation ratio) and LDR (linoleic desaturation ratio) were significantly affected by genotypes and irrigation treatments. The biosynthetic pathway of fatty acids affected by drought stress. This means that linoleic acid increases under withholding irrigation condition, while linolenic acid decreases in such a water deficit stress condition. The highest performance of qualitative and quantitative was detected in HL3721 genotype due to high values of grain yield (3892.45 kg ha−1), oil content (437.05 g kg−1), unsaturated fatty acids (87.63%) and low values of saturated fatty acids (7.98%), and it could be used under withholding irrigation in arid and semi-arid climates. [ABSTRACT FROM AUTHOR]