Pyraclostrobin is one kind of new type methoxy acrylate fungicides that has been widely used in agriculture at present, with a lot of advantages including broad spectrum, high efficiency and high selectivity. In this work, a novel low-level impurity in the pyraclostrobin at about 0.2% was separated and characterized by high-performance liquid chromatography/tandem mass spectrometry (HPLC–MS). Firstly, the impurity was speculated to possess the same skeleton structure as the main product pyraclostrobin while the methyl group on the methyl ester was substituted to be CH2CH2Cl on the basis of the on-line multi-stage mass spectrometric behaviors compared with that of pyraclostrobin. Then the accurate molecular weight and element composition of target impurity was verified to be C20H19Cl2N3O4 by high resolution mass spectrometry. Finally, the proposed structure was further confirmed by the 1H NMR data.