Five heavily biodegraded tar sand bitumens from an oil column were separated into maltene and asphaltene fractions for analysis by negative-ion electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). These bitumens have an identical source, which have experienced a natural sequence of biodegradation. The polar NSO compounds in maltene fractions contain O-1, S1O1, O-3, S1O2, S2O3, O-3, S1O3, O-4, S1O4, N-1, N1O1, N1O3, N1S1, and S-1 classes, while the polar NSO compounds in asphaltene fractions contain O-1, S1O1, S2O1, O-2, S1O2, S2O3, O-3, SO3, S2O3, O-4, S1O4, S2O4, O-5, S1O5, S2O5, O-6, N-1, N1O1, N1O3, N1O3, N1S1, and N-2 classes. Polar NSO compounds with stronger molecular polarity and larger molecular weight are readily fractionated into asphaltene fractions. The 02 class is prevalent in polar NSO compounds of both maltene and asphaltene fractions of all bitumen samples. The NI class in maltene fractions is dominated by compounds with DBE values of 9, 10, 12 and 13, while the NI class in asphaltene fractions is dominated by compounds with a DBE of 15. Most of these N-1 compounds are likely pyrrolic compounds with various numbers of aromatic rings. The biodegradation pathways of nitrogen-containing compounds are also explored in this study. NI species are likely converted to N1O1 and N1O2 species following biodegradation pathways such as ring-opening reaction or carbazole dioxygenase (CARDO) catalytic oxidation reaction. S1O2-5 classes are identified as the dominant sulfur-containing compound classes under negative-ion ESI mode. These classes are considered to contain acid functionalities with higher polarity because the sulfur-containing compounds without oxygen are difficult to analyze by negative-ion ESI in which acids can be ionized by deprotonation. Both progressive oxidation and sulfuration may be involved in the anaerobic biodegradation of sulfur-containing acidic compounds.