To evaluate the gene admixture on the current genetic landscape in Gansu Corridor (GC) in China, the upper part of the ancient Silk Road which connects the Eastern and Central Asia, we examined mitochondrial DNA (mtDNA) polymorphisms of five ethnic populations in this study. Using PCR-RFLP and sequencing, we analyzed mtDNA haplotypes in 242 unrelated samples in three ethnic populations from the GC region and two ethnic populations from the adjacent Xinjiang Uygur Autonomous Region of China. We analyzed the data in comparison with the previously reported data from Eastern, Central and Western Asia and Europe. We found that both European-specific haplogroups and Eastern Asian-specific haplogroups exist in the Gansu Corridor populations, while a modest matrilineal gene flow from Europeans to this region was revealed. The Gansu Corridor populations are genetically located between Eastern Asians and Central Asians, both of who contributed significantly to the maternal lineages of the GC populations. This study made the landscape of the gene flow and admixture along the Silk Road from Europe, through Central Asia, to the upper part of the Silk Road more complete.