Polygonati rhizoma (PR), a traditional medicinal and edible product with various bioactive components (Polygonatum polysaccharides, saponins, phenols, and flavonoids), is widely consumed in China. However, other species with morphological characteristics similar to those of the actual components are being used to replace or adulterate PR, causing issues with quality control and product safety. The morphological similarity of PR and its substitutes makes classic morphological identification challenging. To address this issue, DNA barcoding-based identification using ITS2 and psbA-trnH sequences was applied in this study to evaluate the efficiency and accuracy of this approach in identifying PR samples collected from 39 different regions in China. The identification of PR by this method was confirmed by other methods (phylogeny-based and character-based methods), and all the samples were clearly and accurately distinguished. This study highlights the efficient and accurate nature of DNA barcoding in PR identification. Applying this technique will provide a means to differentiate PR from other altered formulations, thus improving product quality and safety for consumers of PR and its products.