The present study was carried out to evaluate the genetic variations among 20 different populations of Acorus calamus (L.) collected from different natural terrain by RAPD analysis. Total genomic DNAs from individual accessions were isolated from fresh leaf tissue using DNeasy Plant Mini Kit (QIAGEN, USA), according to the manufacturer's instructions and then amplified by eppendorfs Mastercycler gradient themal cycler. Amplified DNA products were quantified by Nanodrop (Genaxy, India) and then subjected to Randomly Amplified Polymorphic DNA (RAPD) analysis. RAPD was used to analyze the genetic variation and relationship among 20 populations of Acorus calamus collected from different part of India, representing four biogeography zones viz., Western Himalaya, Central India, North East India and Eastern Ghats. Twenty five selected RAPD primers produced consistent 235 amplicons ranging in size from 200 bp to 3000 bp; out of which 75 (33.70%) were polymorphic. Similarity indices were calculated using the Dice coefficient of similarity and clustering analysis was performed with the unweighted pair-group method using arithmetic averages (UPGMA) in the SAHN (sequential, agglomerative, hierarchical and nested clustering method) module of NTSYS-PC.The similarity coefficient ranged from 0.91 to 0.98, suggesting that the pronounced low genetic variations exist among populations of A. calamus in Indian germplasm. The cluster analysis indicates the presence of two major groups among the 20 populations of A. calamus regardless of geographical locations. The RAPD analysis indicates the existence of genetic variations in natural populations all the accessions collected from Indian germplasm and they have an extremely low measure of estimated heterozygosity, genetic diversity and polymorphic loci. Genetic variation within a population is homogenous. The pattern of low genetic diversity within the population of A.calamus in Indian germ plasm specifies that they are monoclonal.