Anatomical and TLC study of three Chinese peonies were conducted to make a comparative analysis. Peonies (Paeonia suffruticosa (tree peony), Paeonia lactiflora (Chinese peony) and Paeonia veitchii (Chinese peony)) are traditionally used on the Qinghai-Tibet Plateau in China. Recent studies have shown that the peonies have different pharmacological activities and clinical applications. To distinguish these three species of peonies and ensure the safety and effectiveness in their use, the microscopic characteristics and chromatographic profile of their roots and the corresponding powder were studied. Plant materials sectioned and stained and the raw powder were studied with an optical microscope using standard techniques in microscopy. The results of microscopic features and TLC were described and illustrated. The three species have different microscopic characteristics and TLC profiles, which allow us to distinguish them. In fact, with the help of features semi-quantitative and qualitative, an identification key was developed in our work and illustrated with photos and a table. The aim of our work was to show that the optical microscopy and related techniques provide a achievable practicality, which can be applied without ambiguity to the authentication of species peonies.