Milk contains the milk sugar lactose, which is broken down in the small intestine by the enzyme lactase into easily digestible monosaccharides. Lactase is active in most people until the age of five, after which the expression of the lactase gene, LCT, gradually decreases, which leads to loss of the ability to digest lactose (intolerance), and the consumption of dairy products causes gastrointestinal problems. Consumption of milk and dairy products began with the domestication of livestock in the Neolithic, parallel with the appearance of variants in the regulatory region of the LCT gene, which enable lactase expression and lactose degradation in adulthood. In this paper, the frequencies of alleles and genotypes of variants rs4988235 (-13910C>T) and rs41380347 (-13915T>G) in the Roma populations in Croatia (n = 440) were determined and their association with anthropometric characteristics was examined. The Roma are an example of an isolated population that underwent through several founder effects and is characterized by endogamy. The frequencies of genotypes and alleles of the rs4988235 polymorphism differ significantly between the three Roma populations in Croatia; C allele is more frequent in the populations of Baranja and Zagreb, while T allele associated with lactase persistence, occurs in Međimurje 2x more frequently than in Baranja, and 3x more often than in Zagreb. Polymorphism rs41380347 is monomorphic in the Roma population in Croatia. The examined genetic variants showed no association with anthropometric indicators of nutrition status of Roma. Roma from Međimurje is reproductively isolated from other two investigated populations, and the analysis of the genetic distances of Roma in relation to 20 worldwide populations confirms their Indian origin, as well as sharing gene pool with populations in close geographical proximity.