Abstract Neolithisation was a relatively fast process that affected both the interior and coastal zones of the Iberian Peninsula, but it was also a heterogeneous process that had diverse impacts on genomic and cultural diversity. In the Late Neolithic–Chalcolithic, a change in funerary practices, cultural material and trade networks occurred, and genomic heterogeneity decreased, suggesting human mobility and genetic admixture between different Iberian populations. Dental morphology has emerged as an effective tool for understanding genomic variability and biological affinities among ancient human populations. But, surprisingly, less attention has been paid to the morphological traits of inner dental tissues in Holocene European populations and their utility for the study of population dynamics. We applied 3D geometric morphometric methods on the enamel-dentine junction (EDJ) of the first upper molars to explore the biological affinities of north-eastern Iberian Peninsula populations from the Late Neolithic–Chalcolithic to the Bronze Age. Our results show that the EDJ morphologies of the northern Iberian Peninsula populations were generally homogeneous, indicative of genetic admixture as a result of human mobility and exchange networks. However, differences in the EDJ traits in remains from the Can Sadurní site are indicative of distant biological affinities with nearby populations. Additionally, the hypocone associated dentine area and the position of the trigon dentine horns relative to each other on the occlusal surface best describe the variability found among the samples studied. This study highlights the utility of EDJ morphology as a genetic proxy in Holocene population dynamic studies when paleogenomic studies are absent.