To determine genetic characteristics of the maternal lineage of the Kiso horse based on polymorphisms of the mitochondrial DNA D-loop region, we collected blood samples from 136 Kiso horses, 91% of the entire population, and sequenced 411 bp from 15,437 to 15,847 in the region. First of all, we estimated the demographic history; by searching homology between the obtained and known sequences using Basic Local Alignment Search Tool, by mismatch analysis to evaluate the mutation processes using Arlequin, and by building a phylogenetic tree showing the relationship of the mtDNA haplotypes for 24 horse breeds around the world using Molecular Evolutionary Genetics Analysis softwear. The results suggested that various horses that came to Japan stayed at Kiso region and became ancestors of Kiso horse and also genetically supported the theory that the Kiso horse was historically improved by other Japanese native horse breeds. Next, we analyzed the diversity of current maternal lineage by classifying the resulting sequences, and by calculating the haplotype diversity and nucleotide diversity using Arlequin. Then, we visualized the relationship among haplotypes by a median-joining network using NETWORK 4.6.0.0. The results suggested the diversity of maternal lineage in the Kiso horse was reasonably maintained. Lastly, we predicted future change of the diversity of maternal lineage in Kiso horse by assessing the regional distribution of the acquired haplotypes. The distribution suggested that diversity of maternal lineage would possibly be reducing.