Bulbus Fritillariae (BF), referred to the bulbs of several Fritillaria species (Liliaceae), is a commonly used antitussive and expectorant herb in traditional Chinese medicine (TCM). Due to the complexity of BF botanical origin in the herbal markets, it is urgently needed to develop a reliable method for species identification. Previous studies based on morphological and histological as well as molecular biological techniques have respective limitations. For the purpose of finding a possible discriminant method among Fritillaria species, 27 steroidal alkaloids in 17 Fritillaria species and 12 BF-containing compound formulas were identified and characterized by a high-performance liquid chromatography with mass spectrometry (HPLC–MS) method. The estimated relative composition of steroidal alkaloids was used to carry out a chemotaxonomical study on Fritillaria species by means of hierarchial cluster analysis. In addition, the characteristic occurring patterns of the examined bases were compared in an effort to distinguish the botanical origin of BF-containing compound formulas. The results demonstrated that the qualitative and quantitative differences in steroidal alkaloids were useful not only for chemotaxonomy in some medicinal Fritillaria species but also for species identification in compound formulas. The described method has important implications in quality control of BF-containing TCM preparations, allowing for the prevention of BF confusion, and also revealing the possible presence of adulteration.