Abstract: OBJECTIVE To analyze the quality differences of Anemarrhenae Rhizoma from different areas based on the fingerprint and chemometrics, according to the theoretical basis of the quality marker(Q-Marker) of traditional Chinese medicine, and further combined with network pharmacology, the preliminary prediction analysis of the Q-Marker of Anemarrhenae Rhizoma decoction. METHODS HPLC was used to establish the fingerprints of 20 batches of Anemarrhenae Rhizoma from 6 areas and evaluate their similarity. At the same time, hierarchical cluster analysis(HCA), principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used to screen the main difference components between the groups. Based on the “five principles” of Q-Marker, analyze the different components, and further through the network pharmacology to screen the relevant targets and pathways of the ingredients of Anemarrhenae Rhizoma, construct a “component-target-pathway” network diagram, and predict Anemarrhenae Rhizoma Q-Marker. RESULTS The fingerprint of 20 batches of Anemarrhenae Rhizoma materials were established, 10 common peaks were calibrated, and 7 common peaks were identified, namely, neomangiferin, mangiferin, isomangiferin, timosaponin BⅡ, timosaponin BⅢ, timosaponin Ⅰ, timosaponin AⅢ. Their similarity was >0.934. Twenty batches of samples were clustered into two groups by HCA, PCA and OPLS-DA. Hebei and Inner Mongolia as one category. Shaanxi, Anhui, Shanxi and Shandong as one category. The OPLS-DA screened out neomangiferin, mangiferin, timosaponin AⅢ as the main marker components for the differences of Anemarrhenae Rhizoma. Using network pharmacology analysis to predict that mangiferin, timosaponin BⅡ, timosaponin AⅢ may regulate neural activity ligand-receptor interactions, gap junctions, calcium signaling pathway, cAMP signaling pathway, and cGMP-PKG signaling pathway through NFKB1, TNF, PPP1CC, IL1B, ATP1A3, ATP1A2, ATP1A1 and other targets, played a role in the treatment of neurological diseases, cardiovascular, cerebrovascular diseases and cancer. Combined with Q-Marker theoretical analysis, mangiferin, timosaponin BⅡ, timosaponin AⅢ could be used as potential Q-Markers in Anemarrhenae Rhizoma. CONCLUSION The established fingerprint of Anemarrhenae Rhizoma can directly reflect the chemical composition information of Anemarrhenae Rhizoma from different areas. Combining with network pharmacology, the three chemical components of mangiferin, timosaponin BⅡ, timosaponin AⅢ can be used as the Q-Marker of Anemarrhenae Rhizoma.