This study helps to clarify the debate on the Western and Eastern genetic influences in Central Asia. Thirty-six skeletal remains from Kazakhstan (Central Asia), excavated from different sites dating between the fifteenth century BC to the fifth century AD, have been analysed for the hypervariable control region (HVR-I) and haplogroup diagnostic single nucleotide polymorphisms (SNPs) of the mitochondrial DNA genome. Standard authentication criteria for ancient DNA studies, including multiple extractions, cloning of PCR products and independent replication, have been followed. The distribution of east and west Eurasian lineages through time in the region is concordant with the available archaeological information: prior to the thirteenth-seventh century BC, all Kazakh samples belong to European lineages; while later an arrival of east Eurasian sequences that coexisted with the previous west Eurasian genetic substratum can be detected. The presence of an ancient genetic substratum of European origin in West Asia may be related to the discovery of ancient mummies with European features in Xinjiang and to the existence of an extinct Indo-European language, Tocharian. This study demonstrates the usefulness of the ancient DNA in unravelling complex patterns of past human migrations so as to help decipher the origin of present-day admixed populations.