The GC analysis of fatty acids as their methyl esters derivatives (FAMEs) is an important tool in the characterization of fats in the determination of total fat and trans-fat content in foods. Traditionally, the detailed separation of complex FAMEs requires the use of a long (100 meters or more) capillary column coated with a high polarity cyanopropyl stationary phase to differentiate between the multiple FAME isomers. However, some of the carbon chain lengths usually overlap on the high polarity phase, causing problems in peak identification. Therefore, long analysis time (more than 70 minutes) is required to achieve good FAME separations. The Agilent J&W DB-FastFAME GC column was specifically engineered for the fast separation of FAME mixtures, including some key cis-trans separations. This application shows that the 90m DB-FastFAME GC column can effectively separate 57-component FAME mixture including 37 representative FAMEs and some representative trans FAMEs within 40 minutes, and most of cis-trans isomers can be baselined separated. Analysis of 63-component FAME mixture on DB-FastFAME GC column also demonstrates that DB-FastFAME capillary GC columns can provide rapid analysis and the necessary selectivity to resolve cis-trans pairs in food samples to ensure the food conforms to label requirements.