In order to investigate the population genetic structure of three sympatric Chinese Aquilegia species, we developed a novel set of 12 SSR markers from the EST unigene database. The number of alleles per locus ranged from one to five. The observed heterozygosity (H_o) and the expected heterozygosity (H_e) varied from 0.067 to 1.000 and from 0.064 to 0.791, respectively. Most of the SSR loci in the three Aquilegia species did not significantly deviate from random mating expectation and the Hardy-Weinberg equilibrium (HWE). Because of their high level of polymorphism, the twelve ESTSSR markers will be valuable for mating system, population genetics and conservation studies of Aquilegia resources in the future.