Flavones are significant indicators of quality in TCMs (traditional Chinese medicines) and thus play a significant role in the quality control of TCMs in the pharmaceutical industry. Most flavones in D. nobile Lindl, a traditional Chinese medicine with a long cultivation history and rich sources, have not been identified. This study is aimed at identifying the flavones in D. nobile from different habitats. HPLC coupled with diode array detection (DAD) and HPLC multiple-stage tandem mass spectrometry (HPLC/MSn ) was used to identify the chemical constituents of D. nobile from different habitats, and a method was established to determine the content of vicenin II, violanthin, and isoviolanthin. Hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyse the variations among 26 batches from different habitats. A total of thirty-three flavones were tentatively identified. Twenty-five flavones, previously undescribed in D. nobile, were acylated by p-coumaroyl, feruloyl, sinapoyl or 3-hydroxy-3-methylglutaryl. The D. nobile habitats were distinguished by significant differences in their flavone content. The C-glycosyl flavones were demonstrated to be characteristic compounds for evaluating D. nobile from different habitats. In particular, flavones acylated with 3-hydroxy-3-methylglutaryl (HMG) were specific compounds that were only detected in samples from Yunnan. The results of this study could be used to improve the quality control of D. nobile and could provide references for the identification of acylated C-glycosyl flavones in other natural products. This article is protected by copyright. All rights reserved.