Abstract Polygonati Rhizoma has been a famous traditional Chinese medicine (TCM) for two thousand years. It is increasingly being used not just as a traditional herbal medicine but also as a popular functional food. In this study, qualitative and quantitative analysis of PR from three different origins were initially performed using chemical fingerprint and chemometrics methods. Hierarchical cluster analysis (HCA) and Principal component analysis (PCA) were used to classify 60 PR samples from three different origins. The results revealed that the PR samples fell into three clusters related to the origins. In addition, pairwise comparison of varying PR and obtaining chemical markers between different species through the establishment of partial least squares discriminant analysis. Finally, chemical markers 9,13 and 17 were identified by LC/MS as disporopsin, 5,7‐dihydroxy‐3‐(4′‐hydroxybenzyl)‐6,8‐dimethylchroman‐4‐one and (3R)‐5,7‐dihydroxy‐3‐(4′‐hydroxybenzyl)‐6‐methylchroman‐4‐one or isomer, respectively. In conclusion, these methods can be applied to identify and distinguish the quality of PR with other original plants and provide novel ideas for evaluating herbal products used in TCM.