Recent studies of ancient mitochondrial DNA (mtDNA) lineages have revealed the presence of East Eurasian mtDNA haplogroups in the Central European Neolithic. Here we report the finding of East Eurasian lineages in ancient mtDNA from two Neolithic cemeteries of the North Pontic Region (NPR) in Ukraine. In our study, comprehensive haplotyping information was obtained for 7 out of 18 specimens. Although the majority of identified mtDNA haplogroups belonged to the traditional West Eurasian lineages of H and U, three specimens were determined to belong to the lineages of mtDNA haplogroup C. This find extends the presence of East Eurasian lineages in Neolithic Europe from the Carpathian Mountains to the northern shores of the Black Sea and provides the first genetic account of Neolithic mtDNA lineages from the NPR.