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The macroscopic and microscopic morphologies and indigo and indirubin concentration of the traditional Chinese medicine herbs Isatis indigotica Fort., Polygonum tinctorium Ait., and Baphicacanthus cusia (Nees) Bremek, all commonly known as “daqingye”, were determined and compared. The morphological analyses indicated that I. indigotica has leaves with winged petioles and no glandular hairs or crystals, P. tinctorium has leaves with membranous ocrea and clusters of calcium oxalate, and B. cusia has palisade cells in the mesophyll running over the main vein and single cells containing calcium carbonate crystals. Indigo and indirubin are chemical constituents that have been previously isolated from daqingye and were selected in this study as identification markers for high-performance liquid chromatography analysis due to their pharmacological activities. The chromatographic results showed that indigo and indirubin concentration varied significantly among the three species: high concentration of both indigo and indirubin were observed in I. indigotica, the highest concentration among the three daqingye plants was found in P. tinctorium but with low levels of indirubin, and the concentration of indigo and indirubin was quite low in B. cusia. In summary, three different species commonly known as daqingye were accurately distinguished by morphological observation, internal leaf anatomy analysis, and chromatographic analysis.