The diversity of nine phenotypic traits and ISSR analysis in six natural populations of Paeonia delavayi were studied in this work. The results revealed that abundant variations of all phenotypic traits occurred among and within populations. The coefficient of variation (CV) of nine phenotypic traits ranged from 0.9% to 39.8%, with 18.9% of the mean value. Variation of reproductive organs was larger than that of vegetative ones among populations, while variation of vegetative organs was easily occurred compared with reproductive organs variation within populations. Euclidean clustering analysis showed that the six natural populations could be clustered into four groups. It was indicated that variation of phenotypic traits did not correspond to geography distance. 56 ISSR polymorphic loci were amplified from 180 individuals of 6 natural populations by 10 useful primers. Of them, the value of PPB, H and I was 60.2%, 0.281 and 0.414, respectively, at population level, while the value of H and I was 0.409 and 0.596, respectively at species level. The GST (0.3119) indicated a high degree of genetic differentiation occurred among P. delavayi populations. The results of genetic diversity study, combined with field survey and previous investigations suggest that P. delavayi is not endangered．