The Old Kladruber horse, along with the Lipizzaner horse, Andalusian horse and Lusitano horse, is of the original Italo-Spanish type. The Old Kladruber horse is kept in two colour varieties (grey and black). Because the population is closed, there is a concern about the loss of genetic variation. The genetic diversity and population structure were analysed in the Old Kladruber horse breed based on the pedigree information of animals that were registered in the Studbook to identify factors that may have affected the genetic variability of the breed. Pedigree records collected from 1729 to 2013 contained information on 7971 animals that were used in the analyses. The pedigree depth was up to 33 generations, with an average of 15.1 complete generations. The effective number of founders and ancestors contributing to the current genetic pool was 92.69 and 17.16, respectively. The average values of the inbreeding coefficient were as follows: 13% (with a maximum value of 29%) for the reference population (individuals that can currently take part in reproduction, n=612), 11% for the grey variety (with a maximum value of 25%) and 15% for the black variety (with a maximum value of 29%). The proportion of inbred animals was high (99%). The average rate of inbreeding in the reference population was 1%, 0.8% for the grey variety and 1.1% for the black variety, and the respective estimates of the effective population sizes were 52 for the reference population, 62 for the grey variety and 45 for the black variety. The estimated percentage of genetic diversity lost due to non-random mating within subpopulations and the reference population was 1.0%, 1.0% and 1.2%, respectively. The total loss of genetic diversity in the reference population, in the grey variety and in the black variety was 11%, 13% and 17%, respectively. (C) 2016 Elsevier B.V. All rights reserved.