Abstract: To provide a basis for the establishment of the commodity grade of Astragali Radix (AR), we compared the chemical components and the anti-fatigue effect of different grades of AR. The components of primary metabolites were analyzed by ¹H NMR and the contents of five flavonoids were determined by HPLC-UV with different grades of AR. Fatigue efficacy of different grades of AR was compared. All the procedures were approved by the Laboratory Animal Ethics Committee of the Shanxi University. The results showed that the content of water soluble extracts (WSE) of the Grade Ⅱ AR was the lowest, and 21 compounds were identified through ¹H NMR spectrum. There are 3 components showing a higher content in the Grade-top AR, and 7 components were higher in the Grade-Ⅳ, and 7 other components were higher in the Grade-Ⅱ AR. Total flavonoid content was the highest in Grade-Ⅱ but it was the lowest in the Grade-Ⅳ. Pharmacodynamic results showed that AR could significantly enhance the exhaustion time of rats and improve the biochemical indexes of serum and gastrocnemius muscle, and the best anti-fatigue effect was observed with Grade -Ⅱ AR. Therefore, chemical composition and efficacy index were used to evaluate the quality of different grades of AR, and the quality evaluation approach was established based on chemical and pharmacological effects to provide a scientific basis for the development of AR. The study may provide useful information for construction of the quality grade standard of AR.