The Slovak Pinzgau breed faces the bottleneck effect and the loss of diversity due to unequal use of founders and significant population decline. Further population size reduction can lead to serious problems. High-throughput genotypes of 179 individuals were used to characterise genetic diversity and differentiation of Slovak Pinzgau, Austrian Pinzgau, Cika and Piedmontese cattle by Bayesian clustering algorithm. A gene flow network for the clusters estimated from admixture results was produced. The low estimate of genetic differentiation (FST) in Pinzgau cattle populations indicated that differentiation among these populations is low, particularly owing to a common historical origin and high gene flow. The changes in the log marginal likelihood indicated Austrian Pinzgau as the most similar breed to Slovak Pinzgau. All populations except Piedmontese showed two ways of gene flow among populations indicating that Piedmontese was involved in formation of analysed breeds while these breeds were not involved in creation of Piedmontese. Genetic evaluation represents an important tool in breeding and cattle selection. It is more strategically important than ever to preserve as much of the livestock diversity as possible, to ensure a prompt and proper response to the needs of future generations. The information provided by the fine-scale genetic characterization of this study clearly shows that there is a difference in genetic composition of Slovak and Austrian populations as well as Cika and Piedmontese. Slovak Pinzgau cattle despite its population size has the potential to serve as a basic gene reserve of this breed with the European and World importance.