The objective of the study was to determine the membership probability and level of admixture among Slovak Spotted cattle and historically related breeds (Ayshire, Holstein, Swiss Simmental and Slovak Pinzgau). The analysis was based on the panel of 35 934 SNPs that were used for genotyping of 423 individuals. The optimal number of clusters was estimated in two ways; by analysis of Bayesian information criterion and Bayesian clustering algorithm. The optimal number of clusters ranged from 3 to 5, depending on the applied approach. Subsequently, the population structure was tested by discriminant analysis of principal components (DAPC) and unsupervised Bayesian analysis based on the correlated allele frequencies model. The first discriminant function revealed three genetic clusters in population resulting from the production type and origin of analysed breeds. The unsupervised Bayesian analysis showed similar results, where the highest level of admixture was found between Slovak Pinzgau and Slovak Spotted cattle (0.6%). Despite that, the results of this study clearly showed that the Slovak Spotted cattle is genetically separated from other breeds that were involved in its grading-up process.