Prehistoric Europe experienced a marked cultural and economic shift around 4000 years ago, when the established Neolithic agriculture-based economy was replaced by herding-pastoralist industry. In recent years new data about the genetic structure of human communities living during this transition period began to emerge. At the same time, the genetic identities of the Eneolithic and Early Bronze Age (EBA) inhabitants from a prehistoric cultural crossroad in western North Pontic steppe region remain understudied. This report presents results of the investigation of maternal genetic lineages of individuals buried in kurgans constructed during the Eneolithic-EBA transition in the western part of the North Pontic Region (NPR). Mitochondrial DNA (mtDNA) lineages from the interments belonging to the Eneolithic as well as the EBA cultures such as Yamna (Pit Grave), Catacomb and Babino (Mnogovalikovaya or KMK) were examined. In the 12 successfully haplotyped specimens, 75% of mtDNA lineages consisted of west Eurasian haplogroup U and its U4 and U5 sublineages. Furthermore, we identified a subgroup of east Eurasian haplogroup C in two representatives of the Yamna culture in one of the studied kurgans. Our results indicate the persistence of Mesolithic hunter-gatherer mtDNA lineages in western NPR through the EBA, as well as suggesting a mtDNA lineage continuum connecting the western NPR inhabitants of the Early Metal Ages to the North Pontic Neolithic population groups.