Three genotypes walnut (Juglans regia L.) were investigated during seasons 2019 & 2020 harvests from different selections of trees grown in El Gharbia governorate, Egypt. Physical properties, biochemical and molecular identification of fruit walnut types were examined. Physical properties showed that nut length ranged from 38 to 39.7 mm, nut width from 28.8 to 30.3 mm and nut weight from 11.10 to 12.61g. These genotypes are good quality according to the international walnut descriptor. The chemical analyses showed that total oil ranged from 45.84 to 61.99% while protein ranged from 8.88 to 14.12%. Total carbohydrate was in the range of 44.51-47.35% and the total phenol found between 137.97-147.54mg/g. The Palmitoleic, Oleic and Stearic acids were the main fatty acids in Type-1. Also, the polyunsaturated fatty acid especial linoleic acid was found predominant in Type-2 and Type-3. Stigmasterol (18.21%), Nonadecane (30.07%) and Octacosane (17.17%) were the major in Type-1. On the other hand Eicosane, b-Sitosterol, Pentacosane, and Nonadecane were major sterols in Type -2 while Octadecane was main in Type-3. Based on the DNA rbcL gene sequencing the three genotypes were identified as English walnuts and deposited in the Genbank database under accession numbers (MK002719 and MK002720).