Objective:To distinguish Hedyotis diffusa Willd. and its common countrerfeit, Hedyotis corymbosa. and Hedyotis tenelliflora. by analyzing and comparing their macroscopical identification, microscopic character and HPLC fingerprints. Methods:The features of macroscopical identification, microscopic character including cross-sections of stem, leaf, fruit and seed, and herbal powders were observed in the three samples by traditional methods. The difference of chromatographic peaks among the three samples were also analyzed by HPLC methods.Results:The stems of Hedyotis diffusa Willd. were cylindrical, and the capsules were solitary or double born in the leaf axils, oblate, 2-3 mm in diameter, with a long petiole; the Hedyotis corymbosa. and Hedyotis tenelliflora. were tetragonal, and the Hedyotis corymbosa. was 2-5 capsules born in leaf axils in corymbose inflorescences, globular, 1-1.5 mm in diameter, with a slender petiole; the Hedyotis tenelliflora. were 1-3 capsules clustered in the leaf axils, ovoid with longitudinal ribs around the margin, about 1.5 mm in diameter, without the long petiole, about 1.5 mm in diameter, sessile, the edge of the leaf drying revolute long needle-like. Under the identification, the cross section of the Hedyotis diffusa Willd. stem was almost round, the middle vein of the leaves was protrusion below, the inner pericarp fiber layer consisted of two layers of fiber cells, the surface of the seed coat cells was polygon, and the wall was densely covered with small reddish brown or yellow-brown warty spots. The cross section of the Hedyotis corymbosa. stem was quadrilateral, the surface of the seed coat cell was polygon, the wall was wavy and curved, and there was no warty point on the wall. The middle veins of the Hedyotis tenelliflora. were slightly sunken in the upper part, but not protruding in the lower part; the endocarp fiber layer consisted of 8 to 13 layers of fiber cells. Moreover, the HPLC fingerprint analysis demonstrated substantial dissimilarities in the characteristic peaks of these herbs. Conclusion:The traditional and modern analysis technology show that there are some differences in the characteristics, microscopical cross section, the powder characteristics, which can effectively distinguish the Hedyotis diffusa Willd. and its two local varieties.