The aim of the present study was to analyse the genetic subdivision of the Old Kladruber horse population compared to the historically close Lipizzan breed and to estimate genetic relatedness between them. A set of 13 microsatellites was used for genotyping a total of 270 Old Kladruber horses representing grey and black colour varieties and 418 Lipizzan horses from Slovak and Slovenian studs. The proportion of obtained heterozygosity indicates no major loss of genetic diversity within them. At the individual level across analysed populations, the formation of clusters in respect to breed’s origin and particular studs was observed. The Wright’s FST and genetic distances indicated genetic segregation of both colour varieties at the intraspecific level of the Kladruber breed. Moreover, the membership probability outputs showed that the frequencies of alleles varied across the three main regions represented by both Old Kladruber varieties and Lipizzan, depending on breeding history and strategy of studs.