Ancient DNA technique is a very powerful tool for the studies on past human populations. However, in most cases ancient DNA is extremely degraded into short fragments, and the information is limited because of the damaged state. A large-scale data analysis for human mitochondrial DNA (mtDNA) was carried out to assess validity of the short nucleotide sequence for closely related human populations. We retrieved mtDNA data from the DDBJ/EMBL/GenBank nucleotide sequence database and constructed a data set containing 414 distinct mtDNA types derived from 19 populations of East Eurasia and the surrounding area. A series of new procedures were applied and an mtDNA phylogenetic tree was constructed. Six major star-like clusters were observed in this tree, and the corresponding six radiation groups (I-VI) were characterized. Frequency distributions of each radiation group showed remarkable difference in each geographical area, suggesting that the short mtDNA nucleotide sequences were valuable in analyzing ancient human populations. The efficient procedure for data analysis will enhance the usefulness of ancient DNA data. Additionally, we discuss a possibility of two human migration routes from Africa to East Eurasia based on the mtDNA tree topology and the coalescence times in each radiation group.