Abstract: Surfactin is a class of lipopeptide compounds produced by Bacillus species with significant biocontrol potential. The diversity of its structure and composition directly affects its biological activity. In this study, the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was employed to analyze the compound composition, carbon chain distribution, derivative characteristics, and yield differences of surfactin produced by 18 strains of Bacillus (including closely related genera), aiming to reveal its chemical polymorphism. The results showed that the tested strains could be classified into high-abundance type (≥7 compounds), medium-abundance type (5-6 compounds), and low-abundance type (≤4 compounds) based on the number of surfactin compounds. The carbon chain distribution revealed that C₁₄ and C₁₅ were common to all strains; C₁₃ was present in 11 strains (absent in the remaining 7), while C₁₂ was detected in only two strains; and the long-chain compounds with C₁₆ and above were present only in some strains. The distribution of surfactin derivatives also exhibited strain specificity, with the C₁₅ surfactin derivatives showing the broadest distribution, and only the two strains (Bacillus licheniformis FJAT-8771 and Bacillus alcalophilus FJAT-10014) completely lacking all derivatives. The yield analysis indicated that Bacillus subtilis subsp. inaquosorum FJAT-14251 and Bacillus firmus FJAT-8764 were high-yielding strains, whereas Alkalibacillus haloalkaliphilus FJAT-10023 was a low-yielding strain. The further correlation analysis showed that surfactin production was positively correlated with its compound polymorphism. The high-yield strains generally had the characteristics of high abundance (≥ 7 compounds), broad carbon chain range (including long chains with C₁₆ and above) and multiple derivatives (≥ 3 compounds). This study provided data support for understanding the metabolic diversity in Bacillus, and also provided a reference for screening the superior strains with high production of specific surfactin compounds and for developing the efficient and stable biological pesticides.