The aim of the present study is to document the evolution of the lactase persistence trait in Central Asia, a geographical area that is thought to have been a region of long-term pastoralism. Several ethnic groups co-exist in this area: Indo-Iranian speakers who are traditionally agriculturist (Tajik) and Turkic speakers who used to be nomadic herders (Kazakh, Karakalpak, Kyrgyz, Turkmen). It was recently demonstrated that horse milking practice existed in the Botai culture of Kazakhstan as early as 5,500 BP (Outram et al. 2009). However, the frequency of the lactase persistence trait and its genetic basis in Central Asian populations remain largely unknown. We propose here the first genotype-phenotype study of lactase persistence in Central Asia based on 183 individuals, as well as the estimation of the time of expansion of the lactase-persistence associated polymorphism. Our results show a remarkable genetic-phenotypic correlation, with the causal polymorphism being the same than in Europe (-13.910C>T, rs4988235). The lactase persistence trait is at low frequency in these populations: between 25% and 32% in the Kazakh population (traditionally herders), according to phenotype used, and between 11% and 30% in the Tajiko-Uzbek population (agriculturalists). The difference in lactase persistence between populations, even if small, is significant when using individuals concordant for both excretion of breath hydrogen and the lactose tolerance blood glucose test phenotypes (P = 0.018, 25% for Kazakh vs. 11% for Tajiko-Uzbeks), and the difference in frequency of the -13.910*T allele is almost significant (P = 0.06, 30% for Kazakhs vs. 19% for Tajiko-Uzbeks). Using the surrounding haplotype, we estimate a date of expansion of the T allele around 6,000–12,000 yrs ago, which is consistent with archaeological records for the emergence of agropastoralism and pastoralism in Central Asia.