The latexes of the three Euphorbia species, namely E. antiquorum L., E. nerifolia L., and E. tirucalli L., are highly valued in the Indian system of medicine as purgatives, in addition to their specific and distinct therapeutic activities. In order to distinguish these latexes and develop their diagnostic microscopic and chemical markers, we performed extensive chemical and microscopic studies. The three latexes differ significantly in their microscopic features by exhibiting characteristic starch grain patterns. Although amoebic structures were found to be characteristic of E. antiquorum, dumb-bell and oval structures are characteristic of E. nerifolia and E. tirucalli, respectively. In addition, these latexes showed bone-shaped structures as a common feature, but these differed considerably in their length (10-60, 30-55, and 50-70 μm in length in E. antiquorum, E. nerifolia, and E. tirucalli, respectively). The chemical markers nerifoliene and euphol were found to be common to both E. antiquorum and E. nerifolia, whereas euphol is the only marker for E.tirucalli. A reverse-phase high-performance thin-layer chromatographic (HPTLC) method was developed to distinguish these three latexes and to generate their standard fingerprinting patterns. Most significantly,the markers nerifoliene and euphol could be resolved by RP-18 F254s precoated aluminium plates and the latexes have been quantitatively estimated with respect to these markers. The developed microscopic,chemical and HPTLC patterns can be used to distinguish the three latexes.