The genetic diversity and population structure of seven populations of Sedum alfredii growing in lead/zinc (Pb/Zn) mine spoils or in uncontaminated soils from eastern and southern China were investigated using random amplified polymorphic DNA (RAPD) technology. Four of the sampled sites were heavily contaminated with heavy metals (Zn, Cd, Pb), and extremely high concentrations of Zn, Cd, and Pb were found among these corresponding populations. A significant reduction of genetic diversity was detected in the mining populations. The reduction of genetic diversity could be derived from a bottleneck effect and might also be attributed to the prevalence of vegetative reproduction of the mining populations. Analysis of molecular variance (AMOVA) and the unweighted pair group method with arithmetic mean (UPGMA) tree derived from genetic distances further corroborated that the genetic differentiation between mine populations and uncontaminated populations was significant. Polymorphism with the heavy metal accumulation capability of S. alfredii probably due to the genetic variation among populations and heavy metal contamination could have more impact on the genetic diversity and population structure of S. alfredii populations than geographic distance.