AbstractArchaeogenetic studies have described two main genetic turnover events in prehistoric western Eurasia: one associated with the spread of farming and a sedentary lifestyle starting around 7000–6000 bc (refs. ^1–3) and a second with the expansion of pastoralist groups from the Eurasian steppes starting around 3300 bc (refs. ^4,5). The period between these events saw new economies emerging on the basis of key innovations, including metallurgy, wheel and wagon and horse domestication^6–9. However, what happened between the demise of the Copper Age settlements around 4250 bc and the expansion of pastoralists remains poorly understood. To address this question, we analysed genome-wide data from 135 ancient individuals from the contact zone between southeastern Europe and the northwestern Black Sea region spanning this critical time period. While we observe genetic continuity between Neolithic and Copper Age groups from major sites in the same region, from around 4500 bc on, groups from the northwestern Black Sea region carried varying amounts of mixed ancestries derived from Copper Age groups and those from the forest/steppe zones, indicating genetic and cultural contact over a period of around 1,000 years earlier than anticipated. We propose that the transfer of critical innovations between farmers and transitional foragers/herders from different ecogeographic zones during this early contact was integral to the formation, rise and expansion of pastoralist groups around 3300 bc.