<b><i>Objective:</i></b> The aim of this study was to investigate the frequency of the <i>LCT</i>*<i>-13910C>T</i> polymorphism associated with a high expression of lactase in the small intestine during adulthood, and to infer the lactase persistence and adult-type hypolactasia phenotypes among Euro-Brazilians and Mennonites from South Brazil. <b><i>Materials and Meth</i></b><b><i> ods:</i></b> A sequence-specific PCR method to genotype the <i>LCT</i>*<i>-13910C>T</i> polymorphism in 292 Euro-Brazilians and 151 Mennonites (a group with European ancestry and a long history of endogamy) was developed. Using an exact test of population differentiation, the genotype and allele frequency between these and other Brazilian populations were compared. <b><i>Results:</i></b> The frequency of <i>-13910</i>*<i>T</i> was significantly higher among the Mennonites when compared to the Euro-Brazilian cohort (0.63 vs. 0.33, p < 0.000001). Accordingly, Mennonites had a higher prevalence of the lactase persistence genotype (88.1 vs. 55.5%, p < 0.000001). The distribution of -<i>13910</i>*<i>T</i> differed between Mennonites and all other Brazilian groups (p < 0.0001). The Euro-Brazilians from Curitiba displayed differences when compared to all other Brazilian groups (p < 0.0001), even to Euro-Brazilians from a different geographic region (p = 0.0003), but were similar to those from Porto Alegre (p = 0.2). <b><i>Conclusion:</i></b> Differences in the <i>-13910</i>*<i>T-</i>associated lactase persistence distribution among Euro-Brazilian groups reflect the ancestry and admixture of each particular group and should be considered for adult-type hypolactasia screening.