ObjectiveTo analyze the similarity and variation among different populations of Ocimum sanctum (O. sanctum) by isoperoxidase, isoesterase, acid phosphatase and alkaline phosphatase profile. MethodsThe isolation, separations and staining of the enzymes isoperoxidase, isoesterase, acid phosphatase and alkaline phosphatase were performed as described by Sadasivam and Manickam. ResultsIn isoperoxidase system, the band PRX41 (0.36) was present only in the accession collected from Salem, similar to that band PRX42 showed its presence only in the accession collected from Kolli Hills and PRX51 (0.42) was expressed only in Wyanad accession. The accession collected from Tirunelveli showed its unique banding profile in region ACP23 with 0.18 molecular weight-Rf values in the acid phospahatase system. In esterase system, EST51 and 81 were observed only in the accession collected from Tenkasi. In the alkaline phosphatase enzyme system, only one region (AKP6) of activity with single band (AKP61-0.51) was observed. In this enzyme system, all the selected accession showed their expression. Highest percentage (0.666 7) of similarity was observed between the accessions collected from Tirunelveli and Kolli Hills, Nagercoil and Kolli Hills and Salem and Andaman while the highest percentage (0.800 0) of variation was observed between the accessions collected from Salem and Andaman. The isozyme profile based cladogram showed two clusters, cluster 1 (C1) included the accessions collected from Tenkasi, Tirunelveli, Nagercoil and Kolli Hills and they showed highest percentage of divergence with the other three accessions. The cluster 2 (C2) included the accessions collected from Wyanad, Salem and Andaman. ConclusionsUnique banding profiles of esterase, peroxidase, acid phosphatase and alkaline phospahatase were observed in O. sanctum, which represent the fingerprint of medicinally important plant. Such finger printing is useful in differentiating the species and act as biochemical markers for these species in plant systematic studies in the near future.