A Canadian in situ oilsands bitumen-derived vacuum residue (VR) was subjected to supercritical fluid extraction and fractionation (SFEF) into 13 extractable fractions and an unextractable end-cut and characterized by positive- and negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The results of negative-ion ESI FT-ICR MS showed that the N1 class species was the most abundant and the multifunctional group compounds, such as N1O1, N1O2, N1S1, N1S2, and N2 class species became abundant as the SFEF fraction became heavier. In positive-ion ESI mode, the relative abundance of N1 class species decreased gradually in the heavy SFEF fractions while that of multifunctional group compounds increased. The relative abundance of N4V1O1 increased dramatically in heavy fractions and the end-cut. The distributions of polar heteroatom species of VR derived from oilsands bitumen were similar with those of VR derived from the Venezuela Orinoco extra heavy oil.