The aim of the study was to confirm the presence of previously reported mutations. In addition, we evaluated the single effects of these genotypes on the fatty acid (FA) composition of intramuscular and subcutaneous fat in a total of 602 bulls. Significant effects on FA composition were only determined for the SCD1 and both FASN polymorphisms. The SCD1 c.878C>T genotype was associated with concentrations of stearic, myristoleic and oleic acids. The FASN g.16024G>A and g.17924A>G genotypes did not explain the same part of FA variation and were mainly associated with concentrations of myristic, palmitic, myristoleic and oleic acids. It is concluded that the genetic variations in SCD1 and FASN genes contribute to the variability of FA composition in intramuscular and subcutaneous fat of Fleckvieh cattle.