Acorus calamus L. is a well- known herb for its traditional medicinal and pharmaceutical potentials. The rhizome of the plant is mainly used for medicinal purposes because it yields an essential oil known as "Calamus Oil". The main component of the essential oil is β-asarone. In our present study, 20 different species of Acorus calamus were collected from different parts of South India and North East India. An attempt has been made to characterize all the accessions on the basis of their ploidy level by cytogenetic studies, and determining β- asarone content by HPLC analysis. All the accessions were screened for their ploidy level by staining the root chromosomes at metaphase stage. The plants observed were either diploid or triploid ruling out any tetraploids. HPLC analysis of powdered rhizome extracts for the β- asarone, indicated that it ranged from 2.2% to 7.2%. Our results also have revealed that there is no correlation between the ploidy status and the content of β-asarone. Indian accessions were found to have low to moderate levels of β- asarone content.