<p><i>Daphne genkwa</i> is a deciduous shrub distributed throughout East Asia and has long been used in traditional medicine. Previous phytochemical investigations have shown that <i>D. genkwa</i> is a rich source of bioactive daphnane diterpenoids. In the present study, a comprehensive structural characterization of daphnane diterpenoids in the flowers of <i>D. genkwa</i> was carried out using an LC–MS/MS-based approach. This approach integrated automated annotation program, manual inspection of MS/MS fragmentation pathways, and phytochemical investigation. It led to the structural characterization of 37 daphnane diterpenoids, including nine previously undescribed compounds. These findings demonstrated that <i>D. genkwa</i> flowers contain structurally diverse daphnane diterpenoids, mainly daphnane orthoesters and polyhydroxy daphnanes. Moreover, the diagnostic MS/MS fragmentation and retention time features established in this study provide reliable LC–MS/MS-based criteria for discriminating closely related structural isomers of polyhydroxy daphnanes that differ in their B-ring structures and acylation patterns.</p> Graphical abstract <p></p>