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The endangered species Paeonia rockii is the most important ancestral species of the cultivated tree peonies. These well-known ornamental plants are termed the ‘King of Flowers’ in China. In this study, we investigated the genetic diversity and phylogeographic structure of 335 wild samples from 20 populations throughout the entire distributional range of the species based on three chloroplast DNA sequences (petB–petD, rps16–trnQ and psbA–trnH). At those loci, high levels of genetic differentiation (G ST = 0.94) and low levels of genetic variation (θ = 0.00185) were detected. The intraspecific phylogeny revealed four groups, the western group, the Taibai mountain group, the northern group and the eastern group, which closely coincided with the geographic distribution of the species. A phylogeographic structure of this kind could result from a number of integrated factors, such as allopatric fragmentation, climatic fluctuations, increased abortion and declining germination of seeds, or lack of gene flow among populations, especially across the geographic barrier of the high Qinling Mountains, and it could also result from adaptive evolution. For conservation purposes, each extant population of P. rockii should be recognized as a conservation-significant unit, and a more stringent conservation strategy should incorporate in situ and ex situ methods.