Environmental factors may influence types and contents of active substances. This study investigated the influence of environmental factors on the active substance contents and antioxidant activity ofPotentilla fruticosaL. from different regions of China. Also, HPLC fingerprint similarity analysis (SA) coupled with hierarchical cluster analysis (HCA) and discriminant analysis (DA) were further introduced for the accurate classification and quality assessment ofP. fruticosa. The results showed that altitude was significantly and negatively correlated to the content of tannin (P < 0.05). Annual sunshine duration and altitude were significantly and positively correlated to the flavonoids content, rutin content and antioxidant activity (P < 0.05). Annual mean temperature was significantly and negatively correlated to the content of total phenolics, while altitude was significantly and positively correlated to the content of total phenolics (P < 0.05). Eight samples were unambiguously separated into three groups. Two types of discriminant functions with a 100% discrimination ratio were constructed. All data consistently supported the conclusion thatP. fruticosaproduced from Kangding, Sichuan Province had high quality among all samples, therefore, Kangding in Sichuan Province with favorable environmental conditions is recommended as a preferable production location.