To clarify the origin and genetic diversity of modern horses, mitochondrial DNA (mtDNA) D-loop sequences were generated for 3965 horses from 12 geographical regions. From these sequences, we observed 439 haplotypes defined by 138 polymorphic nucleotide sites. All horses were genetically diverse (HD = 0.973 ± 0.001, π = 0.0243 ± 0.0005), which showed that maternal lineages of the domestic horse are worldwide highly diverse. In general, all 18 haplogroups were presented in the Asian horse. The majority of modern horse sequences belong to haplogroups L, Q, and A. At the same time, 194 archaeological samples from four geographical regions were obtained. Indeed, haplogroup distributions are overlapping in modern and ancient samples, indicating that most haplogroups were already present in ancient times at least in Europe and Asia. The network showed that breeds of Asian and Europe regions overlapped, suggesting that extensive gene flow had occurred between different horse breeds in Asian and European regions.