Background and aims: The yield and quality of medicinal tubers of Rehmannia glutinosa are seriously restricted by replanting problems. However, the origin and identification of the potential allelochemicals associated with this issue have not been well described. Therefore, in a series of related studies, we aimed to identify the key phytotoxic metabolites released from R. glutinosa. Methods: R. glutinosa residues were harvested and extracted for metabolic profiling and bioassay-directed identification. Metabolites in the leaf, tuber, and soil surrounding R. glutinosa roots were profiled using high pressure liquid chromatography coupled to a quadrupole/time of flight mass spectormeter. A Petri dish-filter paper and modified Parker bioassay were used to evaluate the phytotoxicity of plant residues and candidate allelochemicals. Results: Seventeen metabolites specific to R. glutinosa were identified/annotated using non-targeted metabolic profiling combined with known standards. Assay results suggested an iridoid glycoside and several phenylethanoid glycosides as key allelochemicals originating from R. glutinosa. Conclusion: Identification of bioactive metabolites in planta and in the rhizosphere has increased our understanding of the phytochemistry of R. glutinosa. Further evaluation of the soil persistence and ecological functions of these bioactive compounds will be important for determination of the specific mechanisms associated with R. glutinosa replanting issues.