The objective of this study was to determine if variations in single nucleotide polymorphism (SNP) in thioesterase domain of the fatty acid synthase (g.17924 A>G Threonine>Alanine) and in diacylglycerol acyltransferase-1 (g.10433/10434 GC/AA Alanine>Lysine) genes would explain variations in milk fatty acid composition among Holstein dairy cattle. About 200 cows participated in the study. Milk samples were collected monthly throughout the first ten months of lactation and analyzed for milk fatty acid composition by gas chromatography. Blood samples were used to obtain a DNA sample for each animal. Milk from cows with g.17924GG genotype had lower atherogenic index [AI; (12:0 + 4(14:0) + 16:0)/(MUFA + PUFA)] compared with milk from cows of g.17924AG genotype (P=0.007). Likewise, milk from cows with p.232AA genotype had lower AI compared with that from cows of p.232KK genotype (P<0.016). The decrease in AI for cows with g.17924GG and p.232AA genotypes was achieved by the decrease in the concentration of palmitic acid (P=0.06 and P<0.0001, respectively) and by the increase in the concentration of mono- and polyunsaturated fatty acids in milk for both genotypes. The results of this study indicate the potential of using earlier mentioned SNPs as DNA markers to select breeding animals that produce progeny with a healthier milk fatty acid composition.