Fritillaria Cirrhosa Bulbus (known as chuanbeimu in Chinese, FCB) is one of the most used Chinese medicines for lung disease. However, a variety of substitutes have entered the market, with Fritillaria Pallidiflora Bulbus (FPB) being the most common. Due to their similarity in appearance, morphology, and chemical composition but a large price difference, the FCB has frequently been adulterated with the FPB, posing a serious challenge to the distinction and quality of the FCB. Therefore, we aimed to distinguish FCB and FPB based on their main nine isosteroidal alkaloid contents and test the potential of chemometrics as a discrimination approach for evaluating quality. The nine major isosteroidal alkaloids were measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach in 41 batches of FCB and 17 batches of FPB. Additionally, they were categorized and distinguished using the methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA). Quantitative analysis revealed that the nine alkaloids were present in different amounts in the two types of Fritillariae bulbus. In FCB, the highest amount was peimisine (17.92-123.53 μg/g) and the lowest was delavine (0.42-29.18 μg/g), while in FPB, imperialine was higher (78.05-344.09 μg/g), but verticinone and verticine were less than the other seven alkaloids. The FCB and FPB were successfully classified and distinguished by the HCA and PCA. Taken together, the method has a good linear relationship (R > 0.9975). The LOD and LOQ of the nine alkaloids were in the range of 0.0651-0.6510 and 0.1953-1.9531 ng/mL, respectively. The intra- and inter-day precision were shown to be excellent, with relative standard deviations (RSDs) below 1.63% and 2.39%, respectively. The LC-MS/MS method in conjunction with HCA and PCA can effectively differentiate FCB and FPB. It may be a promising strategy for quality evaluation and control at the FCB.