Background: Substitution or adulteration of a particular genuine drug with other species due to demand exceeding the supply of the original species, is rampant in the present trade scenario. As a result, proper authentication of the drug for safe administration as an herbal medicine assumes paramount significance. Aim: Prsniparni, Uraria picta (Jacq.) DC., is one such drug for which three different botanical entities are commonly used as substitutes, namely U. lagopodoides (L.)DC; Desmodium gangeticum (L.) DC., and Pseudarthria viscida (L.) Wight and Arn.; all belonging to the family Fabaceae. The anatomical, histochemical and powder microscopic characters of the four species were compared in the field‑collected samples to validate the quality herbal drug and to find the similarity and dissimilarity of the substitute species. Materials and Methods: Histological and histochemical characters were studied using sectioned materials following standard protocols. Histochemical methods were adopted to localize the presence of the primary metabolites such as starch, lipids, total proteins and amino acids and the secondary metabolites such as volatile oils, resins, tannins, lignin and pectin. Results: The present study shows that the authentic species U. picta and substitute species U. lagopodoides showing higher similarities of 90% based on histology, histochemistry and powder microscopy analysis. Other two candidates, D. gangeticum and P. viscida showing 60 % and 55% similarities, respectively, when compared to U. picta. Thus, the similarity matrix were developed using characters based on anatomical, histochemical and powder microscopy. Conclusion: Ayurvedic texts suggest use of substitute herbs for the rare species. The substitution is proved to be logical by our studies that U. lagopodoides can be used as a substitute species in the place of U. picta under Prsniparni and also the present study validates the genuinity of the drug by anatomical, histochemical as well as powder microscopy characters to quality checking of the raw drug. Key words: Ayurveda, Desmodium gangeticum, histochemistry, powder microscopy, Pseudarthria viscida, similarity matrix, Uraria lagopodoides, Uraria picta