N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagen was successfully used to induce hyper producing neomycin mutants. The neomycin-resistant mutants of Streptomyces albogriseolus, which harvested after treatment with NTG mutagen (100µg/ml) and growing with stress of neomycin (1.75 mg/ml), can produce neomycin with higher efficiency than the wild type strain. The T-40-22 mutant strain was the highest in neomycin production which gave 25 mm inhibition zone by using B. subtilis as the tester organism with 166.67 % relation to the wild type strain after tested of 85 mutants were examined after NTG-mutagenesis. When the above result on inhibition zone was compared with the standard data of neomycin concentrations in relation to inhibition area, the mutant T-40-22 which gave a 25 mm inhibition zone was produced 4.0 mg /mL of neomycin compared with 1.5 mg/mL neomycin of type wild strains and therefore the amount of neomycin production was reached to 2.66 folds of the mother strain production. In addition, the polymerase chain reaction (PCR) technique was applied to examine DNA fingerprint by three random primers on a number of excellent strains for neomycin production to link the genetic background of the tested mutants with the data obtained from the neomycin estimation compared to the wild type strain. Several different DNA fingerprints have been detected after random amplified polymorphic DNA (RAPD) analysis by PCR. Finally, phylogenetic analysis was used to divide the studied mutants into clusters which could reflect the genetic diversity of the new superior neomycin mutants.