It is important to know the relationship between different species in plant breeding. Lilium regale is the key source to breeding modern Trumpet (T) and Oriental-Trumpet (OT) lilies. However, L. sargentiae has not been reported for breeding modern lily cultivars though it has some good horticultural traits. To unveil the relationship between L. regale and L. sargentiae, the present research compared their stomata and photosynthesis, made hybridizations between them using normal pollination and cut style pollination, and analyzed them and their hybrids through fluorescein in situ hybridization (FISH) and random amplified polymorphic DNA (RAPD). The results showed that, except for their stomate size, other characteristics of stomate and photosynthesis were similar; they had indiscriminate FISH karyotypes and were highly crossable; and most PAPD fragment bands were quite similar except that a few bands were species-specific. We concluded that L. regale and L. sargentiae could be considered a biological species though they are two morphologically taxonomic species. Therefore, L. sargentiae could be used in breeding new Trumpet and Oriental-Trumpet lilies as L. regale.