Abstract <p>Modern animal husbandry requires the preservation of genetic diversity to ensure the absence of mutations and diseases caused by the accumulation of unwanted recessive genes, adaptation to climate change, and maintenance of productivity. The study conducted a comparative analysis of the genetic structure of two key breeds of Russian dairy cattle—Holstein and Simmental—using 11 microsatellite markers. From farms of the Ural region, 702 individuals of the Holstein breed and 271 of the Simmental breed were studied. The results showed high genetic diversity in both breeds. The highest observed heterozygosity (<i>H</i>_o) was recorded in the Holstein breed (0.57–0.94); the minimum was in the Simmental breed (0.52–0.89). The number of alleles per locus in cattle of the Holstein and Simmental breeds ranged from 4 to 16, with maximum values in the Simmental breed. Highly polymorphic loci have been identified: <i>TGLA 53</i>, <i>ETH 225</i>, and <i>BM 2113</i>, which are the most informative for monitoring the genetic structure. The level of inbreeding turned out to be low (<i>F</i>_is is close to zero), which confirms the effective management of breeding. However, a deficiency of heterozygotes was found in individual loci, such as <i>SPS 115</i> in the Simmental breed. Comparison with data from other regions has shown that the Ural populations have a wider allelic pool. The practical significance of the work lies in the development of recommendations for minimizing inbreeding and optimizing breeding programs. The study contributes to understanding the importance of preserving the genetic resources of Russian cattle and offers tools to improve the efficiency of breeding.</p>