Bitumen's chemistry often results in complicated intermolecular associations, which are manifested by the diverse microstructures as observed by atomic force microscopy (AFM). These microstructures largely contribute to bitumens' bulk mechanical properties; therefore, it is essential to understand the chemical microstructural mechanical relationships for optimal design of bitumen-related applications. However, the complex nature of bitumen and the various influencing factors often lead to practical challenges in investigation of bitumens' microstructures and their chemical origins. This study aims at addressing some of the main concerns related to AFM characterization of bitumens' microstructures, namely the dependence of bitumens' microstructures on such factors as sample preparation methods, annealing conditions and durations, and chemical composition. Our results suggest that microstructures of bitumen films of a few micrometers or thicker (i.e., the thickness of the asphalt coating layer over the aggregates in asphalt concrete) were comparable regardless of their sample preparation methods, provided that toluene was likely completely removed. Additionally, bitumens annealed at room temperature for over 2 months showed time-dependent microstructures, which correlate well with bitumens' room-temperature steric hardening behavior as verified by other researchers using modulated differential scanning calorimetry. Microstructures of the bitumen films stabilized after different annealing durations depending on the dimensions of the molecular structures and the complexity of the molecular interactions among the multiple phases in each bitumen. Distinctive microstructures were observed for remixed bitumens with increasing asphaltene concentrations. Consistency between our observations and other relevant literature suggests that microstructures observed by AFM are probably not just a surface phenomenon. The above findings provide deeper insights into the establishment of the complicated chemical mechanical relationships for bitumen that pave the path toward tuned bitumen performance.