This study reports the first application of comprehensive two-dimensional gas chromatography coupled to a high-resolution quadrupole time-of-flight mass spectrometer (GC × GC/HRQTOF-MS) for the characterization of naphthenic acid fraction compounds (NAFCs) from the Alberta Oil Sands. High resolution mass spectrometry (HRMS) significantly increased the coverage of NAFCs in the mixture and allowed the differentiation of NAFCs from several chemical classes. It was demonstrated that GC × GC, in combination with the high mass accuracy and precision of the HRQTOF-MS, could distinguish chemical species with the C3 vs SH4 mass split at a much lower resolving power than required with direct infusion experiments. Mass defect plots were useful for visualizing the complex datasets generated by GC × GC/HRQTOF-MS and led to the identification of 1105 chemical species with unique elemental compositions (<5 ppm mass accuracy). Mass defect plots were shown to be a powerful screening tool and enabled the detection of extensive isomer series from the SO2 chemical class, some of which have not been previously reported in oil sands related samples. The GC × GC/HRQTOF-MS approach is expected to improve NAFC monitoring programs since the technique allows the qualitative analysis of individual NAFCs and provides unique fingerprints via isomer distributions which may assist in future fingerprinting studies.