Lilium, a perennial crop with great ornamental, medicinal and edible value, has been frequently used as functional food and medicine. Lilium lancifolium Thunb. (L. lancifolium) and Lilium brownii F.E.Brown var.viridulum Baker (L. brownii) are the most used medicinal species in China. However, the flavor compounds of these two species have not yet been clear. Here, metabolomics and transcriptome analysis were used to reveal the difference of the bitter substances of L. lancifolium and L. brownii. Qualitative results indicated that nine compounds are commonly existed in L. lancifolium and L. brownii, while nine compounds are unique in L. lancifolium and eight compounds are unique in L. brownii. Furthermore, quantitative results revealed that the content of regaloside A in L. lancifolium was nearly 2–7 folds higher than that of L. brownii, and the content of regaloside B in L. lancifolium was about 4–16 folds higher than that of L. brownii. Regaloside C and E were not detected in L. brownii. Transcriptome analysis showed that there were 90 unique genes up-regulated in L. lancifolium samples in the pathway of phenylpropanoid biosynthesis and 75 unique genes up-regulated in L. brownii samples, which could be related to the different content and chemical structure specificity of phenylpropanoid glycerol glucosides in L. lancifolium and L. brownii. The results of our in-deep research provide new insights into the bitter substances of L. lancifolium and L. brownii, and a further consideration for the chemical consistency and quality evaluation for Lilii bulbus.