Aminoglycosides (AMG) are a significant class of antibiotics frequently used with ß-lactams in the management of severe infections brought on by both Gram-negative and Gram-positive bacteria. The clinical efficacy of these antibiotics is currently under jeopardy due to rising Gram-negative bacteria's aminoglycoside resistance. An significant insight into the possible difficulties of treating bacteria comes from the characterization of the gene profiles for antibiotic resistance. E. coli and K. pneumoniae were subjected to a rapid-multiplex-PCR assay to look into the genes producing aminoglycoside-modifying enzymes (AMEs) and assess how common these resistance genes were. The disc diffusion method (Kirby-bauer) was used to assess the antimicrobial susceptibility of 95 bacterial strains against gentamicin, amikacin, neomycin, and tobramycin, with 54 strains of K. pneumoniae and 41 strains of E. coli making up the total. Three distinct sets of primers, aph(3), ant(2), and aac(6), were used in standard multiplex PCR to target AMG resistance genes. Ten distinct resistance patterns for E. coli and K. pneumoniae were found after an analysis of antimicrobial susceptibility tests against AMG for tested bacteria. The most common AME-genes in K. pneumoniae, according to the mPCR, were aac(6) and ant(2) (77.78% for both), followed by aph(3) (48.15%). While among E. coli isolates, aac(6) (75.61%), ant(2) (56.1%), and aph(3) (48.78%) had the highest prevalence of AME-gene resistance. Each isolate of E. coli and K. pneumoniae gained one or more drug-resistant genes, according to the results of the multiplex-PCR. It was hypothesised that the same resistance gene was horizontally spread between other bacterial species.