Tree cultivars (Jupiter, Sejnov, and Elit) of walnut (Juglans regia L.) were collected during the 2004 harvest from Cacak, Central Serbia. The chemical composition, including moisture, total oil content, crude protein, ash, and carbohydrates, was determined. Afterwards, two techniques of oil extraction were implemented: cold pressing extraction and organic solvent extraction. Iodine value, saponification value, acid value, and peroxide value of obtained walnut oils were analyzed. The fatty acid composition of the walnut oils was determined using gas chromatography with flame ionization detector. The oleic acid content of the oils ranged from 15.9–23.7% of the total acids, while linoleic acid content ranged from 57.2–65.1% and the linolenic acid from 9.1–13.6%. The process of oil extraction had no significant effect on content and composition of fatty acids.