The structure of pollen grains is related to the reproductive function of the plants. Here, three-dimensional (3D) refractive index maps were obtained for individual conifer pollen grains using optical diffraction tomography (ODT). The 3D morphological features of pollen grains from pine trees were investigated using measured refractive index maps, in which distinct substructures were clearly distinguished and analyzed. Morphological and physiochemical parameters of the pollen grains were quantified from the obtained refractive index (RI) maps and used to quantitatively study the interspecific differences of pollen grains from different strains. Our results demonstrate that ODT can assess the structure of pollen grains. This label-free and rapid 3D imaging approach may provide a new platform for understanding the physiology of pollen grains.