Petroleum crude is known to contain natural interfacial material (IM) which promotes the formation of stable emulsions. Characterization of the IM is of great interest to the petroleum industry, which allows a better formulation of demulsifiers for the separation of oil-water mixtures in petroleum operations. In this study, IMs in crude oils from Xinjiang, Jianghan, and Daqing, China were separated by the wet silica adsorption method. High-resolution Orbitrap mass spectrometry combined with high energy collisional dissociation was used to reveal the functional groups of separated IMs. The results showed that compound classes of IMs were predominantly O4 (compounds with 4 oxygen atoms in each molecule), O3S1, O4S1, and N1O2. The O4 class species were dicarboxylic acids and their corresponding salts; O3S1 class species were sulfonates; O4S1 class species were either sulfonates or sulfates; and N1O2 class species were amphipathic molecules with both carboxylic and pyridyl functional groups. The composition of IMs are largely different among various crude oils. Most IMs were naturally occurring compounds and some were artificially synthesized chemicals that were introduced in the oil production operation.