In interspecific hybridizations of water lily (Nymphaea), the existence of cross barriers makes it difficult to obtain hybrids and seriously influences the utilization of admirable characters from tropical water lilies. To determine the causes, pollen viability, pistil receptivity, and embryo and endosperm development were investigated in three water lily crosses, including Nymphaea odorata ‘Peter Slocum’ × Nymphaea micranthar (PM), ‘Peter Slocum’ × Nymphaea gigantea (PH), and ‘Peter Slocum’ × Nymphaea colorata (PC). The results indicated that the viability of pollen grains was 17.3% for ‘Peter Slocum’, 19.3% for N. colorata, 10.3% for N. micrantha, and 17.6% for N. gigantea. In the self-pollinated ‘Peter Slocum’, the number of germinated pollen grains on stigmas peaked at 12 hours after pollination (HAP), indicating its good pollen germinability. However, only a few pollen grains germinating on the sigma between 2 and 24 HAP in the crosses of PM, PH, and PC. In addition, a high percentage (81.2%) of normal embryos developed to different stages within 20 d after pollination in the self-pollinated ‘Peter Slocum’. But only 3.5% and 3.7% of normal globular embryos were observed in the PC and PM combinations, respectively. Moreover, no normal embryos were observed in the PH cross. At the same time, no seeds were obtained in PM, PC, and PH crosses. The results suggest that prefertilization barriers existed in the PH cross, whereas pre- and postfertilization barriers existed together in the PC and PM crosses. These may be the main causes resulting in the failure of interspecific hybridizations in water lily.