Hemidesmus indicus (L.) R. Br. (Apocynaceae) root is extensively used in Indian traditional systems due to its biological activities. Decalepis hemiltonii Wight & Arn. is another member from the same family resembling H. indicus and is adulterated in the herbal market. Aim of the study was to compare and evaluate the distinguishing features based on macroscopy, microscopy, powder microscopy, molecular differences in the genomic DNA by RAPD, physiochemical, phytochemical screening, TLC and HPTLC fingerprint profiling of successive extracts. Microscopically cork, cortex, phloem, xylem, medullary rays and pith; powder microscopically size and shape of the cork cells, fibre, fibre tracheids, vessels, xylem parenchyma cells were different from each other. Polymorphism (75.4 %) was found in eight primers out of 16 primers analyzed. The water soluble extractive and the hexane soluble extractive of D. hamiltonii was higher than H. indicus. Tannins, flavonoids, steroids and coumarins were present only in H. indicus and absent in D. hamiltonii. After derivatization, spots at Rf 0.88 (hexane extract), 0.81 (chloroform extract) and 0.55 (ethanol extract) in H. indicus; spots at Rf 0.22, 0.45 (chloroform extract), 0.19, 0.35, 0.58, 0.59 (ethanol extract) in D. hamiltonii were observed. This study will be helpful to find out adulteration of D. hemiltonii in place of H. indicus sold in the crude drug market and in herbal formulations.