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The root bark of tree peony (Paeonia suffruticosa Andr.), also called Coetex Moutan (CM), is a traditional Chinese medicine. The medicinal plant is also well known for its beautiful flowers with hundreds of cultivars. CM from different cultivars or grown in various geographic areas are significantly different in quality and chemical properties. So the aim of this study is to develop specific SSRs by transcriptome analysis to distinguish CM samples from different sources and quantify the contents of paeonol and paeoniflorin to evaluate its chemical qualities. Totally, 6654 SSR markers were developed in the coding regions by transcriptome. 10 pairs of SSRs were found to be consistently polymorphic when applied in all fifty individuals collected from three typical producing areas (Chongqing, Henan, and Anhui province). These CM individuals were originated from two medicinal cultivars of P. suffruticosa and Paeonia ostii T. Hong et J. X. Zhang and horticultural varieties. The genetic results indicated that fifty samples could be clearly divided into three clusters based on genetic distance, in which samples from the same species all gathered together without being affected by geographical origins. The accumulations of two main chemical markers showed great variation among tree peony species or cultivars, in which production area was found as another important factor for this variation except the origin. As a result, the chemical profiles combined with the genetic fingerprint are proved to be effective and could precisely authenticate species and discriminate quality of CM. Image 1 • The identified SSR markers could be effectively applied to reflect the genetic relationship of Cortex Moutan. • The chemical quality of Cortex Moutan from medicinal species are proved better than those from horticultural varieties. • The developed method based on SSR and chemical profiles could distinguish Cortex Moutan from different sources and areas. [ABSTRACT FROM AUTHOR]