Semiaquilegiae Radix has been extensively investigated for its cytotoxic, antitumor, anti-inflammatory, antibacterial, antioxidative, and immunomodulatory properties. The aim of the study was to develop a simple and rapid method for Semiaquilegiae Radix quality control and explore chemical variation within the species using a validated high-performance thin-layer chromatography (HPTLC) metabolomics. Thirty-eight samples were collected from eleven provinces in China. Two HPTLC methods of different polarities were optimized to resolve the major compounds from the sample components. ImageJ software was used to determine and analyze the images of the sample extracts. Chemometric models, including principal component analysis (PCA), hierarchical cluster analysis (HCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to discriminate samples based on the gray value of common bands identified in HPTLC fingerprints. PCA and HCA revealed that samples from the same region have different in chemical compositions. Seven potential chemical markers were screened by variable importance in projection (VIP values > 1.0) of OPLS-DA model and three of them were identified as ferulic acid, griffonilide, and lithospermoside. The HPTLC fingerprint combined with multivariate statistical analyses revealed the differences in chemical components, which enriched the basic studies on the quality control of Semiaquilegiae Radix.