The WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. Associated with the rise in antibiotic resistance is the lack of new antimicrobials. Bacteria have developed many ways by which they become resistant to antimicrobials among those are enzymes. Aminoglycosides play an important role in treatment of serious infections that threat human life caused by Gram-negative bacteria (GNB). In our study, clinical bacterial isolates(210) were collected from different Egyptian hospitals. Eight aminoglycoside antibiotics were used in the present study. The most prevalent pathogen were E. coli (36.66%),E.coli-ESBL(5.23%),Klebsiella spp. (25.33%), Klebsiella spp.-ESBL (1.90%),Pseudomonas spp.(17.61%), Acinetobacter spp. (8.09%),Proteus spp. (2.85%),Enterobacter spp.(1.42%) and (0.47%) for both of Citrobacter sp. and Morganella sp. The most efficiency aminoglycoside antibiotic was Amikin(AK) and the resistance pattern increased over the last years in Egypt. The results of AME-genes indicated that non of aac(3′)-Ia and Rmt(55) genes were detected in any of the thirty Klebsiella spp. and E. coli of Egyptian isolates. The most prevalent AME-genes were aac(3′)-IIa(40%),aac(6′)-Ib (30%) followed by aph(3′)-Ia (23.3%),ant(2″)-Ia(20.0%),aph(3′)-VI(13.3%),and aac(3′)-Ih(6.6%). Also, the results revealed that isolate Klebsiella sp. MAM-16 acquired five AME genes aac(3′)-IIa (accession no. MF495896); acc(3′)-Ih (accession no. MF 495898); aph(3′)-VI(accession no. MF495899); ant(2″)-Ia (accession no. MF495901) and aph(3′)-Ia (accession no. MF495903). E. coli MAM-24 also acquired five AME-genes aac(3′)-IIa (accession no. MF495897); aph (3′)-VI(accession no. MF495900); ant(2″)-Ia (accession no. MF495902); aph(3′)-Ia(accession no. MF495904) and acc(6′)-Ib (accession no. MF495905).