The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this disaccharide. Molecular polymorphism of LAC4 genes encoding β-galactosidase controlling lactose fermentation is not well studied, and the published data concern only a single strain (K. lactis var. lactis NRRL Y-1140) isolated from cream in the United States. We studied β-galactosidase genes in lactose-fermenting К lactis strains isolated from dairy products and natural sources in different regions of the world using molecular karyotyping, Southern hybridization, and sequencing. It was established that the ability to ferment lactose in К. lactis var. lactis dairy yeasts is controlled by at least three polymeric LAC loci with different chromosomal localization: LAC1 (chromosome III), LAC2 (II), and LAC3 (IV). Most of the strains we studied had the LAC2 locus. A comparative analysis of β-galactosidases of the Kluyveromyces genus yeasts and these enzymes from other yeasts was conducted for the first time. Phylogenetic analysis detected significant differences between the LAC4 proteins of yeasts of the Kluyveromyces genus (K. lactis, К. marxianus, К. aestuarii, К. nonfermentans, К. wickerhamii), Scheffersomyces stipitis, Sugiyamaella lignohabitans, and Debaryomyces hansenii. A correlation between β-galactosidase sequences and ecological origin (dairy products and natural sources) of Kluyveromyces strains was found. The group of dairy strains is heterogeneous and includes К. lactis var. lactis and К. marxianus yeasts (99.80-100% similarity), which indicates a common origin of their LAC4 genes. Phylogenetic analysis of β-galactosidases indicates a close genetic relationship of dairy and hospital strains of К. lactis var. lactis and К. marxianus. Clinical isolates are able to ferment lactose and appear to originate from the dairy yeasts.