Genetic resources are a unique and unrecoverable source of genes for further improving the biological and economic potential of production organisms. Preserving agrobiodiversity makes it possible to tackle environmental and climate change, resist against new pathogens and expand breeding possibilities. The aim of the study was to estimate the parameters of genetic diversity of Czech Red cattle and the indigenous Czech Pied cattle and to compare the ancient Czech Red cattle with a revitalized population after a back crossbreeding with Czech Pied cattle. Genomic analysis of SNP markers and genotyping were performed by hybridization of purified DNA to the standard Illumina 50K BeadChip bovine microarrays. Genetic diversity parameters – heterozygosity (H), inbreeding coefficient (FIS), fixation index (FST) and structure of genetic variability (by means of PCA analysis) were estimated by PLINK. The study showed a significant degree of allelic polymorphism (92.6 – 94.32%), but lower values ​​of the average heterozygosity of particular subpopulations (0.362 – 0.377). The zero values ​​of the average inbreeding coefficients can be considered a very positive finding. According to the low values ​​of the fixation index and the result of the PCA analysis, there are no significant genetic differences between the populations of the ancient and revitalized Czech Red cattle. The findings were provided to the National Reference Center for Animal Genetic Resources with regard to the possible use in the targeted protection of endangered gene pools of the indigenous Czech cattle breeds.