Gloriosa superba L. is a highly valued indigenous medicinal plant. It is available throughout India with noticeable variations in morphology and colchicine content. In the present study, morphological, phytochemical, cytological and molecular data are employed to screen the polymorphism, if present, in five different populations of G. superba L. Based on gross morphological traits, the plants can be grouped into two clusters: Amtala (AM) and Baruipur (BR) plants belonging to one cluster, and Siliguri (SG), Darjeeling (DJ) and Sikkim (SK) plants belonging to the other. Colchicine content of the tubers ranged from 0.06% in AM plants to 0.37% in BR plants. Cytological analysis revealed that the SK plants showed 44 chromosomes, i.e. tetraploid status, whereas the other four were diploid. Assessment of the karyomorphology of the four diploid populations indicated a close similarity between AM and BR plants, whereas the SG plants exhibited slightly different karyomorphology and lacked sm-type chromosomes. Random Amplified Polymorphic DNA (RAPD) analysis revealed 76% polymorphism among the populations. Cluster analysis using RAPD resulted in three main cluster groups; BR and SG plants clustered together indicating that they are most closely related when compared with other populations. © 2008 Copyright NIAB.