ABSTRACT: BACKGROUND:Our initial genome-wide association study (GWAS) identified 20 promising candidate genes for milk fatty acid (FA) traits in a Chinese Holstein population, including PRLR, MOGAT1, MINPP1 and CHUK genes. In this study, we performed whether they had significant genetic effects on milk FA traits in Chinese Holstein. RESULTS:We re-sequenced the entire exons and 3000?bp of the 5' and 3' flanking regions, and identified 11 single nucleotide polymorphisms (SNPs), containing four in PRLR, two in MOGAT1, two in MINPP1, and three in CHUK. The SNP-based association analyses showed that all the 11 SNPs were significantly associated with at least one milk FA trait (P?=?0.0456 ~ ?0.05). By the linkage disequilibrium (LD) analyses, we found two, one, one, and one haplotype blocks in PRLR, MOGAT1, MINPP1, and CHUK, respectively, and each haplotype block was significantly associated with at least one milk FA trait (P?=?0.0456 ~ ?A in PRLR, and g.111599360A?>?G and g.111601747?T?>?A in MOGAT1 were predicted to alter the transcription factor binding sites (TFBSs). A missense mutation, g.39115344G?>?A, could change the PRLR protein structure. The g.20966385C?>?G of CHUK varied the binding sequences for microRNAs. Therefore, we deduced the five SNPs as the potential functional mutations. CONCLUSION:In summary, we first detected the genetic effects of PRLR, MOGAT1, MINPP1 and CHUK genes on milk FA traits, and researched the potential functional mutations. These data provided the basis for further investigation on function validation of the four genes in Chinese Holstein.