Ocimum tenuiflorumL., holy basil “Tulsi”, is an important medicinal plant that is being grown and traditionally revered throughout Indian Subcontinent for thousands of years; however, DNA sequence-based genetic diversity of this aromatic herb is not yet known. In this report, we present our studies on the phylogeography of this species usingtrnL-trnFintergenic spacer of plastid genome as the DNA barcode for isolates from Indian subcontinent. Our pairwise distance analyses indicated that genetic heterogeneity of isolates remained quite low, with overall mean nucleotidep-distance of5×10-4. However, our sensitive phylogenetic analysis using maximum likelihood framework was able to reveal subtle intraspecific molecular evolution of this species within the subcontinent. All isolates except that from North-Central India formed a distinct phylogenetic clade, notwithstanding low bootstrap support and collapse of the clade in Bayesian Inference. North-Central isolates occupied more basal position compared to other isolates, which is suggestive of its evolutionarily primitive status. Indian isolates formed a monophyletic and well-supported clade withinO. tenuiflorumclade, which indicates a distinct haplotype. Given the vast geographical area of more than 3 million km2encompassing many exclusive biogeographical and ecological zones, relatively low rate of evolution of this herb at this locus in India is particularly interesting.