Camptothecin (CPT) and its derivatives have attracted worldwide attention due to their notable anticancer activity. However, the growing demand for CPT in the global pharmaceutical industry has caused a severe shortage of CPT-producing plant resources. In this study, phytochemical analysis of Nothapodytes tomentosa resulted in the isolation and identification of CPT (13) and 16 analogues (1-12, 14-17), including a new (1) and five known (9, 10, 12, 15 and 17) CPT analogues with an open E-ring. In view of the potential anticancer activity of CPT analogues with an open E-ring, the fragmentation pathways and mass spectra profiles of these six CPT analogues (1, 9, 10, 12, 15 and 17) were investigated, providing a reference for the rapid detection of these compounds in other plants. Furthermore, based on the fragmentation patterns of CPT (13) and known analogues (2-8, 11, 14, 16, 18-26), the distribution and content of these compounds in different tissues of N. tomentosa, N. nimmoniana, Camptotheca acuminata, and Ophiorrhiza japonica were further studied. Our findings not only provide an alternative plant resource for further expanding the development and utilization of CPT and its analogues, but also lay a foundation for improving the utilization of known CPT-producing plant resources.