Microstructures of bitumen were investigated using atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM), and a chemical characterization was successfully carried out using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The bee structures were observed by AFM, for which a chemical explanation by wax was confirmed by the TOF-SIMS analysis. A tube pattern or worm structures were generated and visualized by ESEM on bitumen surfaces. Chemical differences between the structured and unstructured areas, as well as between different areas of the structure, were observed. A mechanism for the structure formation on bitumen surface during ESEM analysis is suggested.