Pollen morphology on its own and in conjunction with other characteristics has elucidated the origin and evolution of various plant groups. Previous studies of fossil pollen rarely discuss the effects of diagenesis and sample preparation on pollen characteristics, i.e., variability in staining, pollen morphology, and pollen wall ultrastructural characteristics. This paper examines the effect of acetolysis on reflectance and spectral epi-fluorescence measurements. Based on empirical studies, different species under similar experimental conditions display different reflectance values, indicating individual species respond differently to similar post-depositional thermal events. The quantitative pollen fluorescence spectra showed significant variability, but there is an overall increase in the mean wavelength of maximum emission with acetolysis. Increases in these spectral parameters are used to infer thermal maturation and diagenetic pathways in fossil pollen. The paper also discusses observations made on fossil pollen of a known thermal maturity using Pearson's Pollen/Spore Color Standard. Assessment of pollen thermal maturity using this color standard can be an indicator of the quality of morphological and ultrastructural information that can be extracted from fossil pollen. Increasing thermal maturity of pollen may have an effect on staining variability. Based on observations, staining for brightfield 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.