Abstract: Three Epimedium species, E. pubescens Maxim., E. sagittatum (Sieb. & Zucc.) Maxim., and E. wushanense T. S. Ying, which are sympatrically distributed in the western Hubei Province, have been used in traditional Chinese medicine (TCM) for about 2,000 years. Genetic variability and population genetic structure of 11 natural populations of these Epimedium species were investigated using isoelectric focusing in thin-layer polyacrylamide slab gels. Of the 22 enzyme systems prescreened, six coding for 13 loci and 45 alleles were resolved, which were used for analyzing genetic diversity and population structure at both intraspecific and interspecific levels. The results showed that: 1) high levels of genetic diversity were observed in all three species (A = 2.6-3.2, P = 69.2%-84.6%, H_O = 0.274-0.377, H_E = 0.282-0.369), which were higher than that of other herbaceous and animal-pollinated species with similar life-history characteristics; 2) there was significant deviation from Hardy-Weinberg Equilibrium, with one half of the loci showing heterozygote excess and the other homozygote excess, in all populations, suggesting the complicated breeding system ofEpimedium species; 3) the low level of intraspecific and interspecific genetic differentiation (G_ST = 0.0246-0.0409 and 0.0495-0.1213, respectively) indicated a high level of gene flow among populations and close genetic relationship among the three species; and 4) UPGMA cluster analysis further showed that E. pubescens was more closely related to E. sagittatum than to E. wushanense, which was in good agreement with the morphological characters and the recent phylogenetic analysis of these species. On the basis of these results, it was concluded that the mixed breeding system, long-lived perennial life form, ancient evolutionary history, and seed dispersal by ants in Epimedium are responsible for the genetic variation and population structure of these species.