The objective of the study was to determine the genetic distances and admixture in sire lines of Old Kladruber horse (populations in the context of a currently conducted conservation program) base on microsatellite analysis. Basic molecular parameters were estimated. The observed heterozygosity ranged from 0.48 to 0.85. The expected heterozygosity estimated for the sire lines was higher than observed heterozygosity. The minor genetic differences between sire lines of Old Kladruber horse were observed. Differences between the sire lines were identified using discriminant analysis. The first discriminant function separates the individuals of black and grey variants. For the discriminant function, however, the differentiation between sire lines was not so significant. These results showed that the breeding strategy revision is suggested in Old Kladruber horse.