The mitochondrial haplotype U5a1 was identified from an Eneolithic grave associated with the Afanasievo archaeological culture in Bayankhongor Province, Erdenetsogt Township, at the site of Shatar Chuluu. This is the earliest appearance of an mtDNA haplotype associated with modern European populations on the Mongol Steppe. This evidence demonstrations that people with “western” mtDNA lived on the Mongol Steppe east of the Altai Mountains before the Bronze Age and refutes the notion that the Altai Mountains were a substantial barrier to gene flow, and definitively expands the acknowledged range of the Afanasievo archaeological culture.