RATIONALE Chaetoglobosins are a family of macrocyclic polyketide alkaloids. They possess many similar isomers and exhibit a wide range of biological activities. Thus, there is a need for reliable, fast, and low‐cost analysis of this class of compounds. METHODS A series of seven chaetoglobosins from Chaetomium globosum , including two types of isomers, were investigated using electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) in both positive‐ and negative‐ion mode. The identity of major product ions was supported by deuterium‐labeling experiments. RESULTS In positive‐ion mode, the product ion at m/z 130 is the characteristic ion of the indolyl group. A McLafferty rearrangement might play a significant role in the fragmentation of the macrocycle moiety for most chaetoglobosins and produces two series of characteristic product ions, accompanied by neutral losses. The characteristic product ion at m/z 309 in the MS/MS spectrum of chaetoglobosins E indicates the structure of the cyclic olefinic bond in ring B and can be used to distinguish it from the isomers, chaetoglobosins F ex , which has an exocyclic double bond on ring B. In negative‐ion mode, the McLafferty rearrangement has an important role in the fragmentation pattern of the macrocycle. Some high‐abundance radical ions were detected. The radical product ion at m/z 138 might differentiate chaetoglobosins F and penochalasin F, isomers which have very similar structures. CONCLUSIONS In summary, complementary information obtained from fragmentation experiments of [M+H] + and [M–H] – precursor ions is especially valuable for rapid identification of chaetoglobosins. The high‐abundance radical ions in negative‐ion mode are also of scientific interest. Copyright © 2012 John Wiley & Sons, Ltd.