Ocimum species known for its traditional value,which is used as culinary herb and assigned as medicine and spice plant. We used molecular techniques to assess the genetic variability and relatedness of 20 accessions of three germplasm of Ocimum spp. (Ocimum gratissimum, Ocimum sanctum and Ocimum basilicum) collected from different places. DNA was isolated by fixing a sample in alcohol without using liquid nitrogen. Molecular markers were used to assess genetic diversity in basil (Ocimum L. spp., Lamiaceae). Varieties were analyzed through RAPD markers with five Inter-simple sequence repeats (ISSR) primers to determine the extent of molecular characterization. PCR amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multi locus marker system for the study of genetic variation in various organisms. and the result showed various gene expression with the ISSR primer than RAPD markers and we found a maximum number of unique alleles were observed in O. basillicum with ISSR primer (1, 5, 8, 9, 11, 12, 13, 14) which mentioned on primer synthesized table. Among the 15 ISSR primers tested, we have got bands in the size range of 300 bp to 850 bp. Our observations suggested that morphological variability and ISSR analysis could help in identifying genetic variations among different varieties of basil, help in plant improvement and develop a well-organized way to conserve the genetic wealth of basil varieties. DNA based markers like ISSR are more useful than the traditional morphological and biochemical markers. Genetic relationships were examined among thirty germplasm accessions belonging to five Ocimum species using RAPD and ISSR markers.