Recovery of bitumen from oil sands in northern Alberta, Canada, occurs by surface mining or in situ thermal recovery, and both methods produce toxic oil sands process-affected water (OSPW). A new characterization strategy for surface mining OSPW (sm-OSPW) and in situ OSPW (is-OSPW) was achieved by combining liquid chromatography with orbitrap mass spectrometry (MS). In electrospray positive and negative ionization modes (ESI+/ESI–), mass spectral data were acquired with high resolving power (RP > 100 000–190 000) and mass accuracy (<2 ppm). The additional chromatographic resolution allowed for separation of various isomers and interference-free MSn experiments. Overall, ∼3000 elemental compositions were revealed in each OSPW sample, corresponding to a range of heteroatom-containing homologue classes: Ox (where x = 1–6), NOx (where x = 1–4), SOx (where x = 1–4), NO2S, N, and S. Despite similarities between the OSPW samples at the level of heteroatom class, the two samples were very different when considering isomer patterns and double-bond equivalent profiles. The chromatographic separations also allowed for confirmation that, in both OSPW samples, the O2 species detected in ESI– (i.e., naphthenic acids) were chemically distinct from the corresponding O2 species detected in ESI+. In comparison to model compounds, tandem MS spectra of these new O2 species suggested a group of non-acidic compounds with dihydroxy, diketo, or ketohydroxy functionality. In light of the known endocrine-disrupting potential of sm-OSPW, the toxicity of these O2 species deserves attention and the method should be further applied to environmental forensic analysis of water in the region.