Simple SummaryMilk fat content and fatty acid composition are key traits for the dairy industry, as they directly influence consumer acceptance of dairy products and are associated with the chemical-physical characteristics of milk. In order to genetically improve milk fat composition, it is important to understand the biological mechanisms behind the phenotypic variability observed in these traits. In this study, we used a genomic dataset for 6692 animals and over 770,000 genetic markers distributed across the genome. We compared different statistical approaches to better identify the genes associated with milk fatty acid composition in Holstein cattle. Our results suggest that the DGAT1 gene accounts for most of the variability in milk fatty acid composition, and that the PLBD1 and MGST1 genes are important additional candidate genes in Holstein cattle.AbstractThe identification of genomic regions and candidate genes associated with milk fatty acids contributes to better understand the underlying biology of these traits and enables breeders to modify milk fat composition through genetic selection. The main objectives of this study were: (1) to perform genome-wide association analyses for five groups of milk fatty acids in Holstein cattle using a high-density (777K) SNP panel; and (2) to compare the results of GWAS accounting (or not) for the DGAT1 gene effect as a covariate in the statistical model. The five groups of milk fatty acids analyzed were: (1) saturated (SFA); (2) unsaturated (UFA); (3) short-chain (SCFA); (4) medium-chain (MCFA); and (5) long-chain (LCFA) fatty acids. When DGAT1 was not fitted as a covariate in the model, significant SNPs and candidate genes were identified on BTA5, BTA6, BTA14, BTA16, and BTA19. When fitting the DGAT1 gene in the model, only the MGST1 and PLBD1 genes were identified. Thus, this study suggests that the DGAT1 gene accounts for most of the variability in milk fatty acid composition and the PLBD1 and MGST1 genes are important additional candidate genes in Holstein cattle.