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The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the thioesterase (TE) domain of the bovine fatty acid synthase (FASN) gene and to evaluate the extent to which they were associated with beef fatty acid composition. The four exons in FASN that encode for the TE domain were sequenced, and three SNPs, AF285607:g.17924A>G, g.18663T>C and g.18727C>T, were identified. Purebred Angus bulls (n = 331) were classified into three genotype groups, g.17924AA (n = 121), g.17924AG (n = 168) and g.17924GG (n = 42). The g.17924A>G genotype was significantly associated with fatty acid composition of longissimus dorsi muscle of Angus bulls. Cattle with the g.17924GG genotype had lower myristic acid (C14:0; P < 0.0001), palmitic acid (C16:0, P < 0.05) and total saturated fatty acid contents (P < 0.01), greater health index (P < 0.001), oleic acid content (C18:1; P < 0.001) and total monounsaturated fatty acid concentration (P < 0.01) in the total lipids and triacylglycerols fraction than did those with the g.17924AA genotype. Because of the linkage disequilibrium between SNPs g.17924A>G and g.18663T>C, similar significant associations of fatty acid contents with the g.18663T>C genotypes were observed. In conclusion, the SNPs g.17924A>G and g.18663T>C may be used as DNA markers to select breeding stock that have a healthier fatty acid composition.