Ancient Greek colonies were founded by Greek seafarers all along the Mediterranean coast as early as the eighth century bc. Despite extensive archaeological and historical research, the population structure of the inhabitants of Greek colonies and their relationship to indigenous populations are still debated. Here, we perform a biodistance analysis to reconstruct migration and gene flow between the ancient Greek colony of Metaponto (southern Italy) and indigenous groups in the surrounding hinterland (900–250 bc). We collected dental nonmetric trait data of 355 human skeletons from the indigenous Italic sites of Santa Maria d'Anglona, Incoronata and Passo di Giacobbe. This data set is compared with an urban and rural sample of the Greek colony of Metaponto comprising 351 individual dentitions. The R‐matrix approach is used to estimate inter‐population relationships and FST. The resulting kinship coefficients indicate that the three indigenous groups exhibit greater similarity to each other and possess lesser similarity to the two Metapontian samples. Interestingly, the two samples of Metaponto are least similar to each other, although they are geographically very close. The FST estimates confirm this pattern and reveal greater biological variation between the two nearby Metaponto samples (FST = 0.0603) than between the three geographically fairly distant indigenous groups (FST = 0.0389). We conclude that the Greek colony of Metaponto included large numbers of people with diverse geographical origins, whereas the indigenous Italic communities of the surrounding hinterland exhibited high levels of homogeneity and cohesion. Copyright © 2016 John Wiley & Sons, Ltd.