Walnut is one of the most economically significant nut crops. Evaluation of the genetic structure of the domestic walnut gene plasma using modern molecular genetic approaches is a relevant research task. The walnut samples collected by Tsitsin Main Moscow Botanical Garden of Academy of Sciences (MBG RAS) are of particular importance for breeding practice aimed at increasing winter freezing tolerance of plants. The seed material for this collection was introduced from different regions of the former Soviet Union, including Tajikistan, Kyrgyzstan, Ukraine, Belarus, as well as regions of Russia. The MBG RAS collection presents interest as a breeding material for mobilizing the genetic resources and replenishing the gene pool of the South of Russia with new, economically valuable walnut varieties. This work aims to analyze the genetic diversity of a J. regia genotype sample, which includes the most valuable forms from the MBG RAS collection, in order to establish their genetic relationships with samples representing the walnut gene pool of the South of Russia. The genetic analysis of the studied walnut species and varieties was carried out using eight SSR markers: WGA001, WGA376, WGA069, WGA276, WGA009, WGA202, WGA089, and WGA054. The polymorphism of microsatellite DNA markers established during genotyping indicated a high heterogeneity between the MBG RAS walnut sample and genetic resources in other regions. An analysis of genetic relationships using UPGMA and PCoA clustering methods revealed the genetic isolation of most samples in the MBG RAS collection from walnut varieties in the South of Russia. The most genetically distant samples in the MBG RAS collection were found to be 199, 196, 236, 256, 106, and 134. Therefore, these samples should be introduced in the gene pool of North-Caucasus Federal Scientific Center of Horticulture, Viticulture, and Wine-making and Nikitsky Botanical Garden with the purpose of increasing the heterogeneity of their gene pools.