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In Paeonia, hybridization is an important path for both the development of new cultivars and species formation in nature, but the characterization of hybrids has long been a problem. To establish the relationship among Paeonia yananensis, P. jishanensis, and P. rockii, we sampled 159 individuals from 11 populations around the core population of P. yananensis. Samples were subjected to morphological analysis of 22 characters and molecular analysis of three chloroplast DNA (cpDNA) fragments and 14 microsatellites. Paeonia yananensis was distinguishable from the other two species on the basis of morphological characteristics. The phylogenetic tree based on three intergenic spacers in the chloroplast genome (petB-petD, rps16-trnQ, and psbA-trnH) demonstrated that P. yananensis was very closely related to P. jishanensis. However, analysis of 14 microsatellite loci revealed that P. yananensis shared a considerable number of alleles with both P. jishanensis and P. rockii. These results demonstrated that P. yananensis (=P. × yananensis) is of hybrid origin, with P. jishanensis as a maternal parent and P. rockii as a paternal parent. Our success in documenting the hybrid origin of P. yananensis suggests that combined evidence from morphology, maternally inherited cpDNA, and biparentally inherited microsatellites could serve as a powerful tool in hybrid studies. [ABSTRACT FROM AUTHOR]