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Persian walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. Despite the increasing interest in the development of conservation strategies for walnut germplasm, an accurate and full-scale overview of wild genetic resources of J. regia has not been conducted because natural populations are located in regions of Asia historically difficult to access. In this study, we estimated the genetic diversity and spatial genetic structure of 39 autochthonous Persian walnut populations sampled across its Asian range using 14 neutral microsatellite markers. A landscape genetic overlay approach was applied to detect the areas of current reservoirs of walnut genetic diversity in the Asian range and to evaluate the role of landscape in shaping walnut genetic diversity since the Last Glacial Maximum. Although Persian walnut has been highly manipulated by humans over the last 2,000 years, we determined that patches of high genetic diversity still exist in the Caucasus and mountains of Central Asia where J. regia might have survived after Pleistocene glaciations. We detected a clear separation of Persian walnut into four main genetic clusters centered in (1) western Kyrgyzstan, (2) western and south-central Asia, (3) east-central Uzbekistan, and (4) Xinjiang and Shandong provinces (China). Overlay of maps showed a coincidence between groups of walnut populations and potential barriers to gene flow such as the Hindu Kush, Pamir, Tien Shan, and Himalaya mountains and the Karakum, Kyzyl Kum, and Taklamakan deserts. This study claimed the relevance of the preservation of walnut genetic resources in the Asian range.