Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is an important Chinese medicinal herb. Because of overharvesting, the wild populations of this herb have greatly declined and become fragmentized. In this paper, ISSR markers were used to determine the genetic diversity and genetic structure of this variety represented by a total of 153 individuals from three natural populations and three cultivated populations. Fourteen primers produced a total of 251 bands, of which 227 were polymorphic (PPB=90.44%). For the natural populations, the results showed that genetic differentiation was mainly within populations (GST=0.1952), with low genetic diversity at the population level. At the population level, genetic diversity of the cultivated populations was relatively higher than that of the natural populations (PPB=57.24% vs. 53.38%, HE=0.153 vs. 0.151, HO=0.241 vs. 0.235). This pattern can be explained by the recent introduction and artificial selection of cultivars from comparatively wide areas of origin, and subsequent gene flow among populations in cultivation. Although the neighbour-joining cluster analysis seemed to suggest that there was conspicuous genetic differentiation between the natural and cultivated populations, the AMOVA showed that only 4.84% of the total variance existed between groups of natural and cultivated populations, while 67.51% of the variance occurred within populations. In the end, some suggestions for conservation of this important herb are proposed.