Mitochondrial DNA(mtDNA) restriction endonuclease fragment patterns were studied in four different equine populations(Cheju native horses I; Cheju Racing horses, Cheju native horses II; Chejudo Livestock Promotion Institute horses, Tsushima native horses, Thoroughbreds). Total DNA samples from 122 individuals were analyzed with 18 restriction enzymes, Bam HI, EcoRI, HindIII, AvaII, HpaI, BgIII, XbaI, PstI, Pvu II, Sac I, Kpn I, Xho I, AvaI, Hinc II, Bst EII, Not I, Acc I, and Hae II using the horse mtDNA probe. Twelve morphs were observed yielding sixteen different combinations of fragment patterns(mtDNA types) in four populations. The phylogenetic tree constructed from the 12 restriction types by the UPGMA method indicated that mtDNA types in four populations were divided into two clusters. In this tree, the clear separation into the two clusters was observed, namely one with types 1, 6, 3, 8, 9, 2, and 5, and the other one with types 4, 7, 10, 11, and 12. Tsushima native horses were found to be highly homogeneous by the frequencies of mtDNA types. The populations of Cheju native horses I, II, and Thoroughbreds showed polymorphism, while the population of Tsushima native horses was monomorpic(only 1-2-1-1 type), according to the restriction enzymes. Cheju native horses II were shown to be closely related to the Tsushima native horses, and the genetic distance between these two populations was 1.5×10^(-3).