In the present study, each of the 16S ribosomal RNA (rRNA) gene sequences and random amplified polymorphic DNA (RAPD) method were used to confirm the biological classification of three Streptomyces isolates having antiviral activities against Tobacco mosaic virus (TMV). The strongest anti-TMV effect with an inhibition percentage of 97.5 was recorded for S. rochei-IS-02 followed by S. caeaoi-IS-03 (95.5%) and S. aureocirculatus-IS-01 (92.5%) at dilution ½. Lengths of 771, 663 and 693 nts were determined for the partial nucleotide sequences of 16SrRNA gene of S. aureocirculatus-IS-01, S. rochei-IS-02 and S. caeaoi-IS-03, respectively. Sequences producing significant alignments showed identities ranging from 98.41 to 100%. The phylogenetic relationship confirmed the identification of the Streptomyces isolates. A total of 85 DNA fragments (81 polymorphic and 4 monomorphic) were amplified in RAPD assay of the three Streptomyces isolates using eight random primers and contained unique DNA fragments as positive DNA markers. These DNA fragments were distributed as 48, 41 and 42 for the three isolates. The eight primers produced a total amplified DNA fragments of 85 distributed as follows 8, 10, 13, 12, 12, 10, 11 and 9 for the eight oligonucleotide primers, respectively. The similarities between the three Streptomyces species based on RAPD-PCR ranged from 66.332 to 76.158%. The phylogenetic tree showed that S. aureocirculatus-IS-01 and S. rochei-IS-2 were found together in the same cluster, while, S. caeaoi-IS-03 was found in a separate cluster. As a conclusion 16SrRNA and RAPD-PCR methods were confirmed to be more than suitable tools to differentiate between the Streptomyces species.