Based on the analysis of the intron polymorphism of β-tubulin genes, the genetic variability of old Quercus robur L. trees from “Holosiivsky” NPP was investigated. The genotyping of 55 old Q. robur trees was carried out; 40 polymorphic and one monomorphic (about 880 bp) TBR fragments were found. High frequency (70–90%) of occurrence of fragments with an approximate molecular weight of 275, 490, 500, and 1110 bp was observed.The genetic polymorphism of old Q. robur trees was assessed as quite high: РІС is 0.22 – 0.39, the effective number of alleles per locus was 1.174–1.268. The Shannon information index was in the range of 0.204–0.269.The geographical differentiation of the genetic structure of centuries-old oak trees from “Holosiivsky” NPP was not pronounced. The share of inter-selection genetic variability (AMOVA) accounts for about 6% of genetic variability, and the geographic component – about 1%. Around 93% of genetic variability is concentrated on the individual level. Using the ТВР method, we found that Q. robur forest stands do not have a stabilized genetic and visible spatial structure, but at the same time they possess a sufficiently large genetic diversity.