The present study was conducted to identify the association between prolactin and interleukin-8 gene polymorphism and their effects on hormonal concentration, milk yield, and composition traits in Holstein-Friesian cows. Sixty-four Holstein-Friesian milking cows from Gammasa dairy farm close to Mansoura city were categorized into three groups based on milk yield (G1= 20 high, G2= 24 medium and G3= 20 low-producing animals). Serum, blood, and milk samples were collected from the three groups to measure hormone concentration, sequence analysis, and milk components (fat, protein, lactose, and ash). Two novel single nucleotide polymorphisms (SNPs) known as C54G and G75A were detected within the exon three of the prolactin gene. Although high-producing animals with CG prolactin genotype were more frequent in the population (0.62) and significantly showed high serum prolactin hormone concentration, the impact of these SNPs on milk yield traits was statistically non-significant. Moreover, the prolactin C54G showed a significant effect on milk components. Furthermore, a significant association existed between high-producing animals and a higher incidence of mastitis. The results indicated that the population is in Hardy–Weinberg equilibrium, suggesting no change in the distribution of alleles among consequent generations. The CG SNP of the prolactin gene may be a helpful marker for assisted selection programs to improve Holstein-Friesian cattle health and milk composition traits