Probiotics offer a wide range of health benefits to the host. The present paper deals with 16S r-RNA sequence analysis of gut microbial diversity of M. rosenbergii fed with B. licheniformis (MTCC 429; NCBI-GenBank accession number, MK158065 supplemented diet (CFU, 935x10-6). The >10 kb genomic DNA yield ˷1500 pb PCR amplified products against specific 16S r-RNA primers. The aligned sequences of the gut of control prawns showed 1337 bp, 768 bp, 1334 bp, 1419 bp, 1315 bp, 1313 bp, 1466 bp and 1289 bp 16S r-RNA for Pseudomonas sp., Klebsiella oxytoca, Escherichia coli, Bacillus coagulans, Streptococcus thermophilus, Staphylococcus aureus, Citrobacter koseri and Acinetobacter sp., respectively. The gut of experimental prawns showed 1350 bp, 1495 bp, 1464 bp, 1307 bp, 1446 bp, and 1347 bp 16S r-RNA for Bacillus sp., Bacillus licheniformis, Lactobacillus plantarum, Escherichia coli, Streptococcus iniae and Citrobacter sp., respectively. The biochemical tests confirmed that the pathogenic bacteria, like Pseudomonas sp., Klebsiella sp., Staphylococcus sp., and Acinetobacter sp., have competitively been excluded from the gut of experimental prawns due to colony establishment of B. licheniformis and produced good growth [1]. The BLAST of these sequences showed almost 100% similarities with the same species retrieved from the NCBI database. The MAS showed 460 identical amino acids residues, 79 similar amino acids residues and 308 variable amino acids sites for control prawns, and 879 identical amino acids residues, 85 similar amino acids residues and 396 variable amino acids sites for experiment prawns. These sequences have less number of AT biases and more number of GC biases. Overall, the nucleotide divergence and the phylogenetic information calculated were clearly discriminated these bacterial species. Therefore, 16S r-RNA sequencing provides accurate identification of bacterial species.