Allelic and genotypic distribution of polymorphisms in diacylglycerol acyltransferase 1 (DGAT1), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) genes were assessed in 679 Fleckvieh bulls. Single-locus genotype effects and the combined effect of the two polymorphisms within the FASN gene were evaluated for association with the intramuscular fat content and fatty acid profile determined in muscle and subcutaneous fat. The FASN (g.16024G>A) and FASN (g.17924A>G) polymorphisms were significantly associated mainly with C14:0, C16:0, and C18:1 n-9 concentrations as well as with the atherogenic index. The proportion of explained phenotypic variation markedly increased when the effect of a combination of the two polymorphisms within the FASN gene was tested, with the highest values of 8.6% and 14.8%, respectively, observed for C14:0 in muscle and subcutaneous fat. With a focus on improving the fatty acid composition of beef, the results of this study provide valuable information about the markers applicable in marker-assisted selection.