The Old Kladruber horse is the oldest original Czech horse breed – gene resource, with pedigree records spanning three centuries. Because the population is closed, there is a concern about the loss of genetic variation. The genetic diversity within and amongst sire lines was evaluated using microsatellite markers and based on pedigree information. The DNA analysis, covering 16 microsatellite loci, revealed that the genetic diversity (observed heterozygosity = 0.637, genetic diversity = 0.678 and the mean number of alleles = 8.25) for the Old Kladruber horse is consistent with other populations of horses. Inter-line genetic difference in sire lines reached the mean value (the F-ST values ranged from 0.020 to 0.017). Differences amongst the sire lines were identified using genetic distances and principal component analysis. One gene cluster comprised black variation sire lines, while the second cluster included the sire lines of the grey variation. In the subjects monitored, the average inbreeding coefficient of 0.076 was estimated on the basis of pedigree information. The highest mean values of the coefficient of relationship were estimated within the colour variations. When comparing the genetic distance and the average coefficient of relationship, differences were found out. These results are useful for the development of breeding strategies which consider classical horse breeding as well as recent achievements of population and conservation genetics.