In Europe, the Middle Neolithic is characterized by an important diversification of cultures. In northeastern France, the appearance of the Michelsberg culture has been correlated with major cultural changes and interpreted as the result of the settlement of new groups originating from the Paris Basin. This cultural transition has been accompanied by the expansion of particular funerary practices involving inhumations within circular pits and individuals in "non-conventional" positions (deposited in the pits without any particular treatment). If the status of such individuals has been highly debated, the sacrifice hypothesis has been retained for the site of Gougenheim (Alsace). At the regional level, the analysis of the Gougenheim mitochondrial gene pool (SNPs and HVR-I sequence analyses) permitted us to highlight a major genetic break associated with the emergence of the Michelsberg in the region. This genetic discontinuity appeared to be linked to new affinities with farmers from the Paris Basin, correlated to a noticeable hunter-gatherer legacy. All of the evidence gathered supports (i) the occidental origin of the Michelsberg groups and (ii) the potential implication of this migration in the progression of the hunter-gatherer legacy from the Paris Basin to Alsace / Western Germany at the beginning of the Late Neolithic. At the local level, we noted some differences in the maternal gene pool of individuals in "conventional" vs. "non-conventional" positions. The relative genetic isolation of these sub-groups nicely echoes both their social distinction and the hypothesis of sacrifices retained for the site. Our investigation demonstrates that a multi-scale aDNA study of ancient communities offers a unique opportunity to disentangle the complex relationships between cultural and biological evolution.