This study was conducted to evaluate the effect of nitrogen fertilizer with biochar and foliar application of salicylic acid on physiological traits, yield, and fatty acid profile rapeseed cultivars. The treatments were three levels of nitrogen fertilizer along with biochar including 100% N recommended, 10 t ha(-1) biochar + 50% N recommended, and 10 t ha(-1) biochar + 25% N recommended along with foliar application of salicylic acid at two levels of 0 and 0.5 mM and six rapeseed cultivars including Akan, Atora, Danob, Hydromol, Nepton, and Rohan. The results showed that the application of 50% nitrogen + biochar led to increased relative water content (RWC), Chl a, and Chl b contents in leaf by 6, 7.4, and 0.9% compared to 100% N fertilizer, respectively. Foliar spray with salicylic acid increased the seed yield, RWC, Chl a, Chl b, and oleic acid contents by 4.5, 2.9, 7.4, and 2.6% compared with the control, respectively. With foliar application of salicylic acid, the amount of oleic acid increased by 2.6%, and the amounts of palmitic acid, linoleic acid, and linolenic acid fatty acids decreased by 2.3, 6.7, and 0.7%, respectively. In all cultivars, the amount of linoleic acid decreased in the second year compared to the first year, but the amount of oleic acid increased. Among the cultivars, Hydromol showed the higher yield (3667.22 kg ha(-1)), but Rohan represents the minimum value (2401.39 kg ha(-1)). The 50% N + biochar resulted in increased oleic acid (7%) and decreased linoleic acid (21%) compared to 100% N fertilizer. Principal component analysis (PCA) and clustering showed that Akan, Atora, and Danob cultivars were placed in a cluster; Hydromol was classified as a distinguished cluster, while Rohan and Nepton were placed in another cluster. In general, combined of 50% N and 10 t ha(-1) biochar along with foliar application of salicylic acid 0.5 mM in two stages to improve the seed yield quality via increased monounsaturated fatty acid (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) is recommended.