Abstract Introduction Bulbus Fritillaria unibracteata Hsiao et K.C. Hsia is an important traditional Chinese medicine, widely used for the treatment of coughs, phlegm and asthma for thousands of years. Due to an increasing demand in clinic practices, a variety of substitutes have appeared in the market, resulting in a big challenge in the differentiation of bulbus F. unibracteata and its substitutes. Aim To differentiate bulbus F. unibracteata and its substitutes (bulbus Fritillaria ussuriensis Maxim.) based on their main isosteroidal alkaloid contents, and to test the potentiality of chemometrics as a tool for discrimination. Materials and methods The nine isosteroidal alkaloids in 61 batches of Fritillariae bulbus were simultaneously quantitated by liquid chromatography with tandem mass spectrometry (LC–MS/MS) method. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to classify the two kinds of Fritillariae bulbus. Results Quantitative analysis showed that there were differences in the content of the nine alkaloids between two kinds of Fritillariae bulbus. According to the content of nine isosteroidal alkaloids, bulbus of F. unibracteata and F. ussuriensis were successfully distinguished by PCA model. Among these isosteroidal alkaloids, verticine and verticinone may be used as potential chemical markers for the identification and differentiation between the two kinds of Fritillaria bulbus. Conclusion The LC–MS/MS method coupled with PCA would be a powerful strategy to differentiate bulbus F. unibracteata and substitute specimens for quality evaluation and control.