Genetic variation in the Czech Hucul horse population was analyzed using a sequence analysis of the D-loop region of mitochondrial DNA. One hundred and sixty-five Hucul horses were tested. Sequencing of the 700-base pairs fragment of the mitochondrial DNA D-loop region revealed 38 mutation sites representing 14 haplotypes, which were clustered into six haplogroups. The genetic information obtained from the mitochondrial DNA typing is of utmost importance for the future breed-conservation strategies.