Ethyl methane sulfonate (EMS, 200 mM) mutagen was used to obtain superior producing gentamicin mutants Micromonospora echinospora NRRL B-2995 resistant to stress (1000 µg/ml of gentamicin). From 39 mutants, M40-12 was the highest gentamicin producing mutant. The results showed that this mutant gave 25 mm of zone inhibition by using B. subtilis as the tester strain with 178.57% in comparison with the parental strain. The 25 mm inhibition zone of M40-12 mutant equals 1500 µg/mL according to the standard curve of gentamicin; while, parental strain produced 800 µg/mL of gentamicin. So, the gentamicin improvement after EMS-mutagenesis was reached to 1.9 folds. Furthermore, the polymerase chain reaction (PCR) apparatus was used to detect the variabilities between superior gentamicin mutants compared to the parental strain using four random primers. The results indicated different fingerprints using random amplified polymorphic DNA (RAPD) assay by PCR. Also, the phylogenetic analysis was applied to divide the mutants under study into clusters which could reflect the genetic diversity of the new superior gentamicin mutants. Finally, gentamicin optimization by M40-12 mutant and the parental strain demonstrated that the optimum process parameters were a temperature of 34°C, and a pH of 7.5 for gentamicin production by the M40-12 superior mutant. Also, among all the tested organic carbon and nitrogen sources, dextrin and soybean flour were found to be more suitable for high yield of gentamicin production.