The LIPE gene (lipase E, hormone-sensitive type), also known as hormone-sensitive lipase, acts as a primary regulator of lipid metabolism during lactation in cows. We studied a total of two hundred Holstein–Friesian cows and performed sequencing analysis that revealed two synonymous nucleotide changes within the LIPE gene: a transition change, c.276 T > C in exon 2 (g.50631651 T > C; position 351 of GenBank: ON638900) and a transversion change, c.219C > A in exon 6 (g.50635369C > A; position 1070 of GenBank: ON638901). The observed genotypes were TC and CC for the c.276 T > C SNP and CC and CA for the c.219C > A SNP. Notably, the heterozygous TC genotype of the T351C SNP exhibited a significant association with high milk yield. Furthermore, the T351C SNP displayed significant associations with various milk parameters, including temperature, freezing point, density and the percentages of fat, protein, lactose, solids and solids-not-fat, with the homozygous CC genotype showing higher values. The c.219C > A SNP also demonstrated a significant association with milk composition, with heterozygous genotypes (CA) exhibiting higher percentages of fat, protein, and lactose compared to homozygous genotypes (CC). This effect was consistent among both high and low milk producers for fat and lactose percentages, while high milk producers exhibited a higher protein percentage than low milk producers. These findings highlight the importance of considering the detected SNPs in marker-assisted selection and breeding programs for the identification of high milk-producing Holstein–Friesian cows and potentially other breeds. Moreover, this study strongly supports the fundamental role of the LIPE gene in milk production and composition in lactating animals.