Characterization of heteroatom compounds in crude oils is very important in petroleum production and processing. The ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is a powerful MS technique for it but with expensive costs. It is necessary to develop a generally accepted alternative analytical approach to replace FT-ICR MS. In this study, a reversed-phase liquid chromatography (RPLC)-high-resolution (HR) MS method was proposed on an Orbitrap MS with electrospray ionization (ESI). The optimal chromatographic and MS parameters and internal mass calibration method were suggested. The sample complexity was greatly reduced by RPLC separation. Mass resolution (240 K at m/z 200) of Orbitrap MS was enough to obtain an accurate elemental assignment in complex petroleum fractions. False-positive element composition assignment could be easily excluded by the RPLC retention behavior of isomers. The performance of RPLC-Orbitrap MS was evaluated in comparison with direct injection (DI)-FT-ICR MS and DI-Orbitrap MS in positive ion mode for heavy oil analysis. The results showed that heteroatom class species, DBE vs carbon number distributions were very similar between RPLC-Orbitrap MS and DI-FT-ICR MS. The developed methods were further used to analyze petroleum oil fractions with boiling ranges at 180–350 °C, 350–530 °C and >530 °C. Detailed heteroatom class species and corresponding DBE vs carbon number distributions were obtained. This study demonstrated that the RPLC-Orbitrap MS technique could successfully be applied for heteroatom compound characterization in petroleum fractions.