To help plan conservation of the endangered Miyako horse, a biological resource of the Miyako Islands in Japan, we characterized the genetics of the breed by genotyping 32 microsatellites and identifying mitochondrial DNA haplotypes. We also calculated genetic distances between individuals based on the proportion of shared alleles and visualized the genetic relationships with a phylogenetic tree. Two important results were obtained. One is that accurate pedigree registration of the horse by using microsatellites is possible, as the exclusion power of parentage testing is 0.999998. Another is that the current genetic diversity of the horses was clarified. The average number of alleles, observed heterozygosity and expected heterozygosity were 4.2, 0.701 and 0.649, respectively, for the 35 analyzed horses. The probability values for bottleneck models (infinite allele model: 0.00000; stepwise mutation model: 0.00026; and two-phase model: 0.00000) suggested that Miyako horses have experienced a recent genetic bottleneck. Only one mitochondrial haplotype was identified. Consequently, genetic diversity within the population is relatively well-maintained despite a very small population size (41 at the time of the study), and the first priority in conservation of the Miyako horse is to increase the population size.