Abstract <p>The oak grove of Tsitsin Main Botanical Garden has been known since 1731. From 1743 to 1917, it was the property of the Sheremetev Counts. Some information from literary sources suggests that some of the oak plantations are of artificial origin. The objective of this study is to assess the genetic diversity of <i>Q. robur</i> in the oak grove ecosystem on the territory of the Main Botanical Garden of the Russian Academy of Sciences and to determine the genetic relationships with oak trees from the central regions of the European part of Russia. The polymorphism of <i>Q. robur</i> samples was characterized using ISSR analysis. Analysis of the genetic relationships using UPGMA and principal coordinate analysis (PCoA) clustering methods revealed the genetic isolation of the accessions from Vladimir and Ryazan oblasts relative to the accessions from the Main Botanical Garden of the Russian Academy of Sciences, the city of Voronezh, as well as from Moscow, Tver, Tula, and Lipetsk oblasts. The values of genetic polymorphism indices in the population formed in the oak grove ecosystem on the territory of the Main Botanical Garden of the Russian Academy of Sciences are as follows. Effective number of alleles (<i>N</i>_e), 1.480 ± 0.042; genetic diversity index (<i>I</i>), 0.455 ± 0.024; expected heterozygosity (<i>H</i>_e), 0.294 ± 0.020. These parameters can be considered the starting point for genetic monitoring of the population.</p>