A major goal of the Assembling the Fungal Tree of Life project is to create a searchable database <http://aftol.umn.edu> of selected ultrastructural and biochemical characters from published and new data for use in phylogenetic and other analyses. While developing this database such issues as evaluating specimen fixation quality in published micrographs, organizing data to accommodate characters that were dependent on location and developmental stage, and requiring accountability of data contributors were addressed. Character states for three traits, septal pore apparatus, nuclear division and spindle pole body cycle, are illustrated, and character states are resolved with maximum parsimony and plotted on a summary cladogram of known phylogenetic relationships of the Fungi. The analysis illustrates the inherent phylogenetic signal of these characters, the paucity of comparable characters and character states in subcellular studies and the challenges in establishing a comprehensive structural and biochemical database of the Fungi.