In order to clarify the morphological characteristics of the Nymphaea pollen, the morphological characteristics of 18 species or cultivars of Nymphaea were observed by scanning electron microscopy in this study. The pollen morphological characteristics and relationships between subgenera and species were analyzed, and the pollen viability of the N. ‘Peter Slocum’ was investigated by in vitro germination method. The study provides a basis for selecting parents for hybrid breeding of Nymphaea. The results showed that: (1) The pollen of 18 species or cultivars in Nymphaea were all single grains, and the morphology was nearly spheroid or oblate spheroid. Among them, the pollen grains of N. gigantea were the largest, volume index was 30.3, and the pollen grains of N. lotus were the smallest, volume index was 19.2. (2) There were many types of patterns in water lily pollen, including checkered shape, small granular, nodular, small cylindrical, long strip. (3) According to the cluster analysis, the 18 species or cultivars of Nymphaea were divided into four subgenera, among which the Nymphaea and Brachyceras were most closely related. (4) The pollen germination rate of N. ‘Peter Slocum’ was more than 95%. It was believed that the pollen morphological characteristics of Nymphaea, especially the type of pollen germination gully and the patterns, could be used as the key evidence for the classification of Nymphaea species, the relationship between subgenera and species, and the law of Nymphaea.