This article offers a new look at the stratigraphy and chronology of Mesolithic and Neolithic deposits at Lepenski Vir, particularly based on newly available Accelerator Mass Spectrometry (AMS) dating and aDNA genomic evidence. It focuses on a detailed analysis of several key contexts for which new radiocarbon dates are available while at the same time reviewing taphonomic and age-offset problems when dating human remains and other materials affected by the aquatic reservoir effect in the Danube Gorges area. The robust chronological evidence as well as available stratigraphic data overwhelmingly show that the start of the main and iconic phase of the occupation of this site, represented by the architecture of trapezoidal buildings and sculpted sandstone boulders, should unequivocally be dated to the period of the Mesolithic-Neolithic transition in the last two centuries of the seventh millennium cal BC. At this time, local forager populations of distinct hunter-gatherer genetic ancestry came into contact and mixed with incoming Neolithic, farming populations of north-western Anatolian genetic ancestry, based on the available genomic data but also supported by studies of material culture traditions. The article deals directly with the recent criticism of this chrono-stratigraphic model for Lepenski Vir.