The aim of this work was to gather information about the origin and genetic characterization of the Central European Hucul horse based on 71 horses using 17 microsatellites and the D-loop region of mtDNA. Their genetic relationship to the Polish Konik (N = 7), German (N = 4) and Hungarian wild Przewalski horses (N = 4) and 200 horse sequences from GenBank was also analysed. Both microsatellite and mtDNA analysis showed a high genetic variation in the Hucul. A total of 130 alleles were detected, the mean number of observed alleles per microsatellite was 7.647, and the number of effective alleles was 4.401. The average observed and expected heterozygosity were 0.706 and 0.747, respectively. The high heterozygosity values and Wright's fixation index (FIS) (−0.128) indicated a low level of inbreeding, low or no selection pressure, and large number of alleles. mtDNA analysis revealed 18 haplotypes for the Hucul population with a total of 23 variable sites. Haplotype and nucleotide diversities were 0.935 ± 0.011 and 0.022 ± 0.012, respectively. Neutrality tests (Tajima's D and Fu's Fs) were non-significant, and mismatch distribution was ragged, indicating that the Hucul population is in genetic equilibrium. The most frequent mtDNA D-loop region belonged to haplogroup A (48%), which was also present in Przewalski Wild horse samples, while Polish Konik samples belonged to three haplotypes and C, F, and G haplogroups. Large and significant pairwise ΦST values along with a small number of common haplotypes indicated a low level of gene flow and lack of genetic structure among the three studied breeds (Hucul, Konik, and Przewalski Wild horse). The present work contributes to our knowledge of the genetic diversity of the Hucul horse and helps to define its genetic conservation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 54–65.