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Genome selection (GS) technology is an important means to improve the genetic improvement of dairy cows, and the mining and application of functional genes and loci for important traits is one of the important bases for accelerating genetic improvement. Our previous study found that the apolipoprotein A5 (<i>APOA5</i>) and AKT serine/threonine kinase 3 (<i>AKT3</i>) genes were differentially expressed in the liver tissue of Chinese Holstein cows at different lactation stages and influenced milk component synthesis and metabolism, so we considered these two genes as the candidates affecting milk production traits. In this study, we found in total six single nucleotide polymorphisms (SNPs), three in <i>APOA5</i> and three in <i>AKT3</i>. Subsequent association analysis showed that the six SNPs were significantly associated with milk yield, fat yield, protein yield, or fat percentage (<i>p</i> ≤ 0.05). Three SNPs in <i>APOA5</i> formed a haplotype block, which was found to be significantly associated with milk yield, fat yield, and protein yield (<i>p</i> ≤ 0.05). In addition, four SNPs were proposed to be functional mutations affecting the milk production phenotype, of which three, 15:g.27446527C>T and 15:g.27447741A>G in <i>APOA5</i> and 16:g.33367767T>C in <i>AKT3</i>, might change the transcription factor binding sites (TFBSs), and one is a missense mutation, 15:g.27445825T>C in <i>APOA5</i>, which could alter the secondary structure and stability of mRNA and protein. In summary, we demonstrated the genetic effects of <i>APOA5</i> and <i>AKT3</i> on milk production traits, and the valuable SNPs could be used as available genetic markers for dairy cattle’s GS.