Studies of genetic markers of uniparental inheritance, such as mitochondrial DNA have been used successfully to study the settlement, history and origins of human populations. In the Chilean population, most published articles on the subject have been carried out with populations from southern Chile, leaving a gap on our knowledge about northern populations. In order to delve into population dynamics of the coast and interior valleys of the Antofagasta region, we analyzed 207 individuals from the cities of Antofagasta and Calama, to determine the frequency of mitochondrial haplogroups in each one. The results show a significant difference between the coast and the valleys; despite their geographical proximity, the genetic differences between them suggest they can be considered different population units. These differences probably result from the social and cultural patterns of these populations in the past, which, due to their kinship networks, were developing different ways of life over time: the coast and the puna lifestyles, leaving a biological imprint that lasts to this day.