Uruguay has one of the highest per capita milk intakes worldwide, even with a limited supply of lactose-free products; furthermore, the admixed nature of its population is well known, and various frequencies of lactase persistence (LP) are observed in the source populations. We aimed to contribute to the understanding of the relation between allelic variants associated with LP, milk consumption, digestive symptoms, and genetic ancestry in the Uruguayan population. Samples of saliva or peripheral blood were collected from 190 unrelated individuals from two regions of Uruguay, genotypes for polymorphic sites in a fragment within the LCT enhancer were determined and allelic frequencies calculated in all of them. Data were collected on frequency of milk and dairy consumption and self-reported symptoms in a subsample of 153 individuals. Biparental and maternal ancestry was determined by analyzing individual ancestry markers and mitochondrial DNA. Twenty-nine percentage of individuals reported symptoms attributed to the ingestion of fresh milk, with abdominal pain, bloating and flatulence being the most frequent. European LP-associated allele T-13910 showed a frequency of 33%, while other LP-associated alleles like G-13915 and T-14011 were observed in very low frequencies. Associations between self-reported symptoms, fresh milk intake, and C/T-13910 genotype were statistically significant. No evidence of association between genetic ancestry and C/T-13910 was found, although individuals carrying one T-13910 allele appeared to have more European ancestry. In conclusion, the main polymorphism capable of predicting lactose intolerance in Uruguayans is C/T-13910, although more studies are required to unravel the relation between genotype and lactase activity, especially in heterozygotes.