This study aimed to investigate the effects of grafting on the fatty acid profile and some physicochemical properties of watermelon seed and seed oil. The ‘Crimson Tide’ cultivar was used as the scion while two wild watermelon (Citrullus lanatus var. citroides (A1 and A2)), one Lagenaria siceraria (A3) and one Cucurbita maxima Duchesne x Cucurbita moschata Duchesne (A4) were used as rootstocks. The use of rootstock significantly influenced the fatty acid profile and the physical parameters of seeds and seed oils. The highest linoleic acid ratio was found in the seed oil from A1 and A2, the oil from A3 had the highest oleic acid ratio. The results showed that the content and acid value in seed oils were improved, and that total phenolic compounds and antioxidant activity of both seed and oil were decreased by grafting. Wild rootstocks can be used in watermelon cultivation to obtain a watermelon seed which is rich in linoleic acid.