Persian walnut (Juglans regia L.) has unique nutritional properties, with substantial benefits to human health. This research mainly aimed to evaluate biochemically twelve selected genotypes from the Hamadan province of Iran. The amounts of kernel oil, protein, and various fatty acids (FAs) were recorded. The kernel biochemical analysis showed that protein and oil content varied from 10.75 to 17.49% and from 66.17 to 74.10%, respectively. Based on the results, the content of FAs was determined according to the oil analysis of genotypes. The range of various amounts of the major kernel oil FAs comprised palmitic acid (6.16-8.57%), stearic acid (2.21-3.77%), palmitoleic acid (0.00-0.54%), oleic acid (11.83-39.28%), linoleic acid (37.91-63.82%), and linolenic acid (7.78- 14.89%). It was observed that the range of changes in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) were between 8.73-11.26, 18.66-46.74 and 51.05-77.70%, respectively. Also, changes in total phenol content in the studied genotypes ranged between 42-60 mg gallic acid (eq) g-1 dry weight of the extract (GAES g-1 mg). In conclusion, the studied genotypes showed superiority in the most important walnut breeding traits and showed suitable biochemical compounds such as PUFAs composition with a good nutritional value.