Bacillus species are aerobic spore-forming bacteria that are known pose a significant risk to human health. Even though numerous methods for classifying Bacillus cereus and Bacillus thuringiensis have been reported, it remains very difficult to accurately and rapidly distinguish these Bacillus species since they are genetically very similar. In this study, we described to identify biomarkers for classifying B. cereus and B. thuringiensis by metabolomic profiling using ultraperformance liquid chromatography–tandem mass spectrometry. Established method were provided for classifying B. thuringiensis and B. cereus led to the identification of 92 and 38 potential candidate biomarkers in cellular and media fractions, respectively. Among them, one particular ion (1193.676 m/z) had accurately classified for three food samples with the suspected contamination of B. cereus or B. thuringiensis. Our study proposed a new biomarker to rapidly distinguish B. cereus group members and might be very useful in the food, cosmetic, and agricultural industries.

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