Separation of fatty acids as methyl ester (FAME) derivatives has been carried out using short and highly polar capillary column developed for fast gas-liquid chromatography (GLC) applications. The GLC parameters have been optimized in order to achieve separation of FAME ranging from 4:0 (butyric acid) to 24:1 in less than 5 min. Milk fat that has by far the most complex fatty acid composition among edible fats and oils has been used to optimize the method. The volume of the oven has been reduced in order to allow for a heating rate of 120 degrees C/min and to rapidly cool-down to the initial temperature (50 degrees C) of the GLC program. The GLC conditions developed are not suitable to achieve separation of positional and geometrical isomers of octadecenoic acid but are useful to perform separation of major fatty acids in milk fat. The conditions developed could be used to analyze edible fats and oils or biological samples such as plasma or red blood cell lipids. The results confirmed that short and highly polar fast columns operating under optimal conditions could be used to separate the fatty acids in various matrices.