Comparative morphology among species of the genus Calostoma, including C. cinnabarina, C. ravenelii, and C. japonicum, was investigated by scanning electron microscopy and atomic force microscopy. Spore morphology of C. cinnabarina and C. ravenelii showed no dramatic differences by light microcopy and scanning electron microscopy. To differentiate these species, atomic force microscopy was employed. Quantitative analysis of the surface roughness of basidiospores revealed subtle differences in height fluctuation at the nanometer scale between the species of Calostoma. Basidiospores of C. cinnabarina had a relatively rougher surface than those of C. ravenelii at $2.0{ imes}2.0;{mu}m^2$ scan areas.