The ability of humans to digest the milk component lactose after weaning requires persistent production of the lactose-converting enzyme lactase. Genetic variation in the promoter of the lactase gene (LCT) is known to be associated with lactase production and is therefore a genetic determinant for either lactase deficiency or lactase persistence during adulthood. Large differences in this genetic trait exist between populations in Africa and the Middle-East on the one hand, and European populations on the other; this is thought to be due to evolutionary pressures exerted by consumption of dairy products in Neolithic populations in Europe. In this study, we have investigated lactase persistence of 26 out of 46 individuals from Late Neolithic through analysis of ancient South-West European DNA samples, obtained from two burials in the Basque Country originating from 5000 to 4500 YBP. This investigation revealed that these populations had an average frequency of lactase persistence of 27%, much lower than in the modern Basque population, which is compatible with the concept that Neolithic and post-Neolithic evolutionary pressures by cattle domestication and consumption of dairy products led to high lactase persistence in Southern European populations. Given the heterogeneity in the frequency of the lactase persistence allele in ancient Europe, we suggest that in Southern Europe the selective advantage of lactose assimilation in adulthood most likely took place from standing population variation, after cattle domestication, at a post-Neolithic time when fresh milk consumption was already fully adopted as a consequence of a cultural influence.