The aim of the research was to evaluate the genetic structure of Ukrainian Holstein cattle (n = 150) of the state enterprise Chayka (Kiev region, Ukraine) on the basis of the analysis of 12 microsatellite DNA loci and 3 QTL genes: k-casein (CSN3), prolactin (PRL), and fatty acid synthase (FASN). A total of 93 allelic variants were identified, and the largest number of alleles were found for TGLA53, TGLA122 and TGLA227 loci, the mean PIC = 0.693, and the heterozygosity index for all loci was 0.7314. The greatest number of effective alleles per locus were found for the BM1824 locus. Based on association analyses, complex genotypes were established for the three genes (CSN3, PRL, FASN). It was found that the AAPRL AACSN3 AAFASN genotype determined the highest milk yield (8351 kg/lactation) and the protein content in milk (3.10%), whereas the AAPRL ABCSN3 GGFASN genotype determined the highest level of fat content in milk (3.80%).