Oil–sand naphthenic acids (NAs) are organic wastes produced during the oil–sand digestion and extraction processes and are very difficult to separate and analyze as individual components due to their complex compositions. A comprehensive two-dimensional gas chromatography/time of flight mass spectrometry (GC × GC/TOF–MS) system was applied for the characterization of two commercial mixtures of naphthenic acids (Fluka and Acros) and a naphthenic acid sample extracted from the Syncrude tailings. Contour plots of chromatographic distributions of different Z homologous series of the Fluka, Acros and Syncrude NAs were constructed using fragment ions that were characteristic of the NA's molecular structures. Well-ordered patterns were observed for NAs of Z = 0 and −2 which corresponded to acyclic acids and monocyclic acids, respectively. For NAs of Z = −4, −6, and −8, specific zones were observed which would allow the pattern recognition of these NAs obtained from different origins. As expected, gas chromatographic retention times increase with the number of the carbons and the number of rings in the molecules. Little signal was obtained for NAs with Z numbers of −10, or lower. Deconvoluted mass spectra of various NA isomers were derived from the reconstructed GC × GC chromatogram, permitting detailed structural elucidations for NAs in the future. The current study demonstrated that the combination of GC × GC and the TOF–MS is a powerful to identify origins of the NAs in an effective manner. GC × GC/TOF–MS alone, however, may not be enough to characterize each individual isomer in a complex mixture such as NAs. The use of mass deconvolution software followed by library search have thus become necessary to separate and study the mass spectrum of each individual NA component, allowing a detailed identification of the toxic components within the NAs mixture.