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The objective of this study was to investigate the physicochemical characteristics of oil extracted from genetically modified cottonseed cultivar (CN-CO2) and local non-genetically modified cultivar (Hamid). The physicochemical characteristics determined were specific gravity, refractive index, color, iodine value, saponification value, unsaponifiable matter, free fatty acids, acid value and peroxide value. In addition the fatty acids composition for the two cultivars was determined. Cultivar Hamid contained 22.7% oil, while CN-CO2 contained 15.5%. The two oil samples showed significantly (P≤0.05) different specific gravity; CN-CO2 had higher value (0.92) than that of Hamid (0.908); similar refractive index with a mean value of 1.458 as well as slightly different color. Hamid and CN-CO2 cultivar showed similar peroxide values (6.2 and 6.8 meq/kg, respectively); but significantly (P≤0.05) different iodine value (100.02 and 103.22 g⁄100g, respectively), saponification value (186.7 and 200.5 mg KOH⁄g, respectively), free fatty acids (2.2 and 1.9%, respectively), acid value (4.4 and 3.7 mg KOH⁄g, respectively) and unsaponifiable matter (2.1 and 1.2%, respectively). The percentage of numbers of saturated and unsaturated fatty acids was equal (50% each) in Hamid but in CN-CO2, they were 40.1% and 59.9%, respectively. Furthermore, three fatty acids vis butyric, caprylic and margaric appeared in Hamid but not found in CN-CO2. Conversely, capric, heptacosylic and arachidonic acids detected in CN-CO2 but not in Hamid. No significant difference in the remaining fatty acids, were observed between the two cultivars. In conclusion; the two cultivars differed in oil content and most of its studied characteristics.