Venezuela Orinoco extra-heavy-crude-oil-derived vacuum resid (VR) was subjected to supercritical fluid extraction and fractionation (SFEF) to prepare multiple narrow fractions. The SFEF fractions were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with various ionization techniques, including positive-ion electrospray ionization (ESI), negative-ion ESI, positive-ion atmospheric pressure photoionization (APPI), and sulfur methylation followed by positive-ion ESI. The results showed that the SFEF separates the VR species by their molecular weights and degrees of molecular condensation. The mass ranges of compounds determined by various ionization techniques were comparable. The FT-ICR MS data were in agreement with the elemental analysis and molecular weight determined by gel permeation chromatography (GPC) and vapor pressure osmometry (VPO) for the extractable fractions. The molecular compositions of SFEF fractions determined by FT-ICR MS provide important clues for the understanding of the molecular composition for the unextractable end-cut (asphaltenes). Each ionization technique favors identification of certain compounds in heavy petroleum fractions and discriminates against others. APPI allows for a general overview of species present in heavy petroleum fractions, because of its ability to ionize a wide range of species. ESI is more selective toward polar species. A thorough characterization of species in heavy petroleum fractions cannot be achieved by using an ionization technique; however, it can be performed by combining various ionization techniques.