Pollen morphology and pollen morphology in conjunction with other characteristics have elucidated the origin and evolution of various plant groups. Many studies of fossil pollen rarely discuss the effects of diagenesis and sample preparation on pollen characteristics. This paper examines the relationship of diagenesis, sample preparation (e.g., acetolysis), and staining techniques on the preservation and interpretation of pollen characteristics viewed with light, scanning and transmission electron microscopy. Based on empirical studies, different species under similar experimental conditions have different reflectance values, indicating the individual species respond differently to diagenetic events. The quantitative pollen fluorescence spectra showed significant variability, but an overall increase in the mean wavelength of maximum emission. Increases in these spectral parameters are used to infer thermal maturation and diagenetic pathways in fossil pollen. Assessing the thermal maturity of a dispersed or in situ pollen using Pearson’s Pollen/Spore Color Standard can be an indicator of the quality of morphological and ultrastructural information that can be extricated from fossil pollen. Staining for light or electron microscopy in fossil pollen, although useful for improving contrast in the specimen, must be used with caution when interpreting pollen wall structure. Although single fossil pollen investigations are useful, replication of these studies on similar or the same specimens from the same locality or various localities will collectively provide more information for elucidation of the morphology and ultrastructure of the once living pollen, and is helpful in sorting out characteristics that may be artifacts from post depositional diagenesis.