Polygala tenuifolia Willd. is an important protected species used in traditional Chinese medicine. In the present study, amplified fragment length polymorphism (AFLP) markers were employed to characterize the genetic diversity in wild and cultivated P. tenuifolia populations. Twelve primer combinations of AFLP produced 310 unambiguous and repetitious bands. Among these bands, 261 (84.2%) were polymorphic. The genetic diversity was high at the species level: percentage of polymorphic loci (PPL)=84.2%, Nei's gene diversity (h)=0.3296 and Shannon's information index (I)=0.4822. Between the two populations, the genetic differentiation of 0.1250 was low and the gene flow was relatively high, at 3.4989. The wild population (PPL=81.9%, h=0.3154, I=0.4635) showed a higher genetic diversity level than the cultivated population (PPL=63.9%, h=0.2507, I=0.3688). The results suggest that the major factors threatening the persistence of P. tenuifolia resources are ecological and human factors rather than genetic. These results will assist with the design of conservation and management programs, such as in natural habitat conservation, setting the excavation time interval for resource regeneration and the substitution of cultivated for wild plants.