The paper presents data on the assessment of the genetic diversity of five subpopulations of the European bison (Bison bonasus) in Belarus — from the National Park “Belovezhskaya Pushcha”, the National Park “Pripyatsky”, the Osipovichi district (Mogilev area), SEI “Berezinsky Biosphere Reserve” and the Grodno region. In general, the work includes 30 samples of muscle tissue from the collection of Gene bank of wild fauna in SSPA “SPC NAS of Belarus on Bioresources” (Minsk, Belarus). Microsatellites were used as markers to assess genetic diversity, structure, and search for signs of a sharp decline in the size of bison subpopulations in the past. A total of 11 microsatellite markers were used, recommended by the Food and Agriculture Organization of the United Nations for cattle research. The analysis of B. bonasus subpopulation from the NP “Pripyatsky” showed signs of passing through the genetic bottleneck. All studied subpopulations are characterized by a similarly low genetic diversity level in all analyzed indicators (mean number of alleles, allelic diversity, observed and expected heterozygosity). The expected heterozygosity (He) for the three subpopulations from the NP “Belovezhskaya Pushcha”, the NP “Pripyatsky” and from the Osipovichi district ranged from 0.37 to 0.39. For the studied subpopulations, the values of the fixation index were negative. The assessment of the presence of genetic structuring between the subpopulations of bison from the NP “Belovezhskaya Pushcha”, the NP “Pripyatsky” and from the Osipovichi district based on the values of such indexes as Fst and DJost which showed no signs of genetic differentiation, which is also confirmed by principal coordinates analysis (PCoA). The European bison conservation in Belarus has required tremendous efforts in the past. So far, even though the impressively large population size reached in Belarus, B. bonasus status still should not be considered as stable, which is closely linked to aspects of its overall low genetic diversity. Our research confirmed the low genetic variability of Belarusian subpopulations. Therefore, the more extensive research concentrated on identifying genetic diversity is necessary to ensure the beneficial control of gene flow and register a potential correlation of unfavorable gene variants with possible inbreeding depression. These attempts are required to lay the groundwork for the management and protection of the European bison in Belarus.