The study aimed to determine the differentiation of biodiesel prepared from regular and aged soybean oils, through a comprehensive two-dimensional gas chromatography, combined with a main component analysis (PCA), as well as to identify the compounds present in both raw materials. In order to evaluate the samples in the 2D system, three secondary stationary phases were established: SBL-IL61, Supelcowax and HP-50. Results showed that only polar secondary coating, SBL-IL61, was able to perceive a crucial difference between the samples. The largest difference was observed in the identification of the highest concentration of polyunsaturated fatty acid methyl ester (FAME) in aged soybean biodiesel, such as C20: 4n-6, C20: 5n-3, C20: 3 and C21: 5. These compounds are quite prone to oxidation, which would lead to devaluation of biodiesel as a final product, thus may be used as markers for biodiesel degradation. In consequence, an analytical technique that provides increased detection of these biodiesel quality-dependent compounds is recommended.