We investigated the occurrence and intra-allelic variability of the T(-13910) variant located upstream of the lactase gene in 965 individuals from 20 different locations of Italy and Greece. The T(-13910) frequency ranges from 0.072 (Sardinia) to 0.237 (North-East Italy), with a statistically significant difference between North-East Italians and other Italian populations. The comparison of the lactose tolerance predicted by T(-13910) and that assessed by other studies using physiological tests shows a one-way statistically significant discrepancy that could be due to sampling differences. However, the possible role of other genetic factors underlying lactase persistence is worth exploring. The time of the most recent common ancestor and departures from neutrality of the T(-13910) allele were assessed using three microsatellite loci. Time estimates were found to be congruent with the appearance of dairy farming in Southern Europe and the occurrence of a single introgression event. Robust signals of selection can be observed in North-East Italy only. We discuss the possible role of cultural traits and genetic history in determining these observed micro-evolutionary patterns.