Acorus calamus is a well-known medicinal and aromatic plant used for many industrial and pharmaceutical purposes. In our current study, twenty different accessions of Acorus calamus collected from North-East India and South India have been studied and an attempt has been made to classify them according to their ploidy level by cytogenetic analysis and to correlate them via PCR-RFLP technique for the Non- Transcribed Spacer (NTS) region of the 5S-rRNA gene. β-asarone which is the major phyto-chemical found in Acorus sp. when present in higher concentrations was found to be carcinogenic. The scope of this study is to identify diploid Acorus calamus accessions as they are reported to have less concentration of β-asarone from those of triploids. Cytoyping was performed by staining of root metaphase chromosomes. For PCR-RFLP analysis, the NTS region of 5S rRNA gene was amplified by using primers, designed from the coding regions of 5S rRNA gene. The PCR products were digested with EcoRI restriction enzyme. Triploid accessions showed restriction digestion at 586 bp resulting in an additional fragment of 127 bp, which is absent in diploids. Ten diploid accessions and 10 triploid accessions were identified by cytogenetic technique and PCR-RFLP method. The sequences of triploid and diploid were analyzed using bioinformatics tools.