Sesquiterpene pyridine alkaloids (SPAs) are a large group of macrocyclic dilactones formed by dihydro-beta-agarofuran sesquiterpenes and pyridine dicarboxylic acids, which have aroused widespread interest in the field of medicine due to their significant biological activities. In order to achieve accurate and rapid characterization of SPAs, previous studies have systematically investigated their fragmentation patterns in MS2, but there are still some problems that need to be resolved, such as distinction between wilfordate (W)- and evoninate (E)-subtypes, as well as determination of the substituent position. In this paper, a comparative analysis of W- and E-subtypes of SPAs was carried out by a product ion scanning-multistage fragmentation (PIS-MSn) strategy using ultra-high-performance liquid chromatography/linear ion trap quadrupole/orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS). The precursor ions for MSn were selected from the ions related with pyridine dicarboxylic acid moiety in MS2 spectrum, such as the ions at m/z 206, 178, and 160. The results showed that the product ions at m/z 104 or 117 were produced in the MS4 spectra of W-subtype SPAs ([M+H](+) > m/z 160 > m/z 132 > m/z 104 or 117). The ion at m/z 104 was produced when acetyloxyl group was present at C-5, whereas the ion at m/z 117 was produced when hydeoxyl group was present at C-5. Neither of the above characteristic ions were produced in the MS4 spectra of E-subtype SPAs. The fragmentation mechanism was also deduced. In addition, 13 SPAs were identified from the root of Tripterygium wilfordii, including 1 unknown compound using the MS/MS fragmentation pattern. The findings in this study enable the differentiation between W- and E-subtypes of SPAs and, to a certain extent, solve the problem of substituent position, which is of great significance for the accurate and in-depth identification of SPAs.