Abstract: Approximately 0.5 g of the powdered Anisodus acutangulus medicinal herbs after being sieved was taken and placed in a triangular flask, and 50 mL of 80% (volume fraction) ethanol solution containing 0.2% (volume fraction) sulfuric acid was added. The mixture was extracted by reflux at 85 ℃ for 1 h, and after cooling, the mixture was shaken well and filtered through a 0.45 μm organic phase filter membrane. The filtrate was determined by high performance liquid chromatography, and gradient elution was performed by mixed solutions composed of 0.05% (volume fraction) phosphoric acid solution and methanol at different volume ratios as mobile phase. The chromatograms were recorded and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 edition) was used for establishment of fingerprint of Anisodus acutangulus medicinal herbs. The different markers that had great influence on the quality of Anisodus acutangulus medicinal herbs were screened by principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA). The 3 alkaloid components, including hyoscyamine, anisodamine and scopolamine, were determined. As shown by the results, a total of 17 common peaks were identified by fingerprints of 20 batches of Anisodus acutangulus medicinal herbs from different origins, and the similarities were in the range of 0.756-0.988. The results of PCA and OPLS-DA were consistent, and 20 batches of Anisodus acutangulus medicinal herbs from different origins were divided into 3 categories. Four different markers were screened, including camphor alkaloid, and the 3 alkaloid components mentioned above. Taking hyoscyamine, anisodamine, and scopolamine with relatively high contents as the detection index components, the determination results of the 3 detection index components in 20 batches of Anisodus acutangulus medicinal herbs from different origins were analyzed by cluster heatmap. As shown by the results, among the Anisodus acutangulus medicinal herbs from different origins, the quality from Yunnan Province was the best, while the quality from Guizhou Province was the worst.