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The aim of the study is to identify changes in genetic diversity at the BoLA-DRB3 gene exon 2 of Ukrainian Black-and-White dairy cows on the example of a breeding herd where, over the past decade, targeted selection for increasing Holstein bloodlines has been carried out to improve milk production. Changes in genetic diversity were determined by comparing the biodiversity of the experimental herd with Holstein populations. In three stages, blood samples were obtained from 391 cows of the Ukrainian Black-and-White dairy breed from the Kozatska Dolyna 2006 breeding farm, Khmelnytsky oblast, Ukraine. The allelic spectrum of the BoLA-DRB3 gene exon 2 was determined by PCR–RFLP. The study was carried out in stages: in 2008–2010 in the first population (maternal herd, n = 162), in 2014–2017 in the second population (daughter herd, n = 123), and in 2019–2022 in the third population (granddaughter herd, n = 106). For comparison, the study includes previously published data on Holsteins: United States (n = 1100), Canada (n = 835), Chile (n = 113), Argentina (n = 424), Bolivia (n = 159), Paraguay (n = 139), and Peru (n = 133). The high polymorphism of the BoLA-DRB3 exon 2 gene makes it convenient for analyzing the genetic diversity of cattle. The assessment by Simpson and Berger-Parker indices shows that the measured herd of Ukrainian Black-and-White dairy breed has more genetic diversity at the BoLA-DRB3 gene than the Holstein populations. The allelic structure of the studied herds, evaluated by the Pielu index and Buzas and Gibson evenness, is more uniform. Significant genetic diversity of the Ukrainian Black-and-White dairy breed is confirmed by the Shannon index and the effective number of alleles. Their values are significantly higher than those for all the considered Holstein cattle populations. In the time trend, a decrease in genetic diversity at the BoLA-DRB3 gene was found in Ukrainian Black-and-White dairy cows compared to Holstein cows. The linear forecast for the future period showed that the level of its genetic biodiversity will decrease at the current level of use of Holstein genetic material. [ABSTRACT FROM AUTHOR]