Walnut (Juglans regia L.) is exceptionally widespread nut tree in the world and grow best in Azad Jammu and Kashmir and northern areas of Pakistan is expected to have considerable inter  and  intra-species  genetic variability  due  to  adaptation  to  the  various  environmental  conditions. We focused to evaluate the genetic characterization of sixty four walnut cultivars including their genetic relationship and distance collected from sixteen selected sites of Neelum valley Azad Jammu and Kashmir in Himalayan region of Pakistan. 35 decamer primers were used which based on discernable bands yielded markable amplification patterns. Cluster analysis and principal component analysis (PCA) separated the sixty four cultivars into six main groups whose alterations were related to their pedigree from each other. In our study we found 44% cultivars had marker alleles associated with the presence of WGA gene. 30% had at VAM gene. 18% had at JM gene. Six markers did not show polymorphism (were not validated). Frequencies of JM and VAM genes need to be improved in future walnut cultivars. Advanced walnut cultivars may routinely be investigated for the presence or absence of their special variety genes by the use of DNA markers. Kagazi akhrot need to be pyramided through MAS to extend the genetic base of future varieties. Moreover, fieldwork study revealed that local walnut germplasm are being eroded from some regions due to urbanization, agriculture, grazing, wood harvesting and natural calamities (earth quake and land sliding) that depicted genetic drift. The insightful difference among walnut accessions confirmed that they did not develop locally but were introduced from abroad germplasm whereas, close relationship showed that they are probably from same population with common ancestry.