Fourteen effective polymorphic ISSR primers were tested and selected. The genetic diversity in wild Staphylea bumalda of Anhui province was analysed based on the selected primers. A total of 143 polymorphic loci were amplified, showing effective number of alleles per locus (n_e) of 1.271, expected heterozygosity (H_e) of 0.165, and Shannon’s information index (I) of 0.257 at the population level, and n_e = 1.341, H_e = 0.227, I = 0.370 at the species level. The genetic differentiation among populations (G_st) stood at 0.271. Preliminary results revealed the genetic diversity in wild S. bumaldas, and indicated the applicability and reliability of ISSRs to the investigation of genetic diversity in S. bumaldas. The present study will provide support for further research of multi-population genetic diversity in S. bumaldas and conservation of its genetic resources in future.