Heavy oil molecular mixtures were investigated on the basis of single molecules resolved by atomic force microscopy. The eight different samples analyzed include asphaltenes and other heavy oil fractions of different geographic/geologic origin and processing steps applied. The collected AFM data of individual molecules provide information about the molecular geometry, aromaticity, the content of nonhexagonal rings, typical types and locations of heterocycles, occurrence, length and connectivity of alkyl side chains, and ratio of archipelago- vs island-type architectures. Common and distinguishing structural motifs for the different samples could be identified. The measured size distributions and the degree of unsaturation by scanning probe microscopy is consistent with mass spectrometry data presented herein. The results obtained reveal the complexity, properties and specifics of heavy oil fractions with implications for upstream oil production and downstream oil processing. Moreover, the identified molecular structures form a basis for modeling geochemical oil formation processes.