Abstract Aminoglycosides are used in treating a wide range of infections caused by Gram-positive and Gram-negative bacteria; however, aminoglycoside resistance is common and occurs by several mechanisms. Among these mechanisms is bacterial rRNA methylation by the 16S rRNA methyl transferase (16S-RMTase) enzymes; but data about the spread of this mechanism in Egypt are scarce. Cephalosporins are the most commonly used antimicrobial agents in Egypt; therefore, this study was conducted to determine the frequency of 16S-RMTase among third generation cephalosporin-resistant clinical isolates in Egypt. One hundred and twenty three cephalosporin resistant Gram-negative clinical isolates were screened for aminoglycosides resistance by the Kirby Bauer disk diffusion method and tested for possible production of 16S-RMTase. PCR testing and sequencing were used to confirm the presence of 16S-RMTase and the associated antimicrobial resistance determinants, as well as the genetic region surrounding the armA gene. Out of 123 isolates, 66 (53.66%) were resistant to at least one aminoglycoside antibiotic. Only one Escherichia coli isolate (E9ECMO) which was totally resistant to all tested aminoglycosides, was confirmed to have the armA gene in association with bla TEM-1, bla CTX-M-15, bla CTX-M-14 and aac(6)-Ib genes. The armA gene was found to be carried on a large A/C plasmid. Genetic mapping of the armA surrounding region revealed, for the first time, the association of armA with aac(6)-Ib on the same transposon. In conclusion, the isolation frequency of 16S-RMTase was low among the tested aminoglycosideresistant clinical samples. However, a novel composite transposon has been detected conferring high-level aminoglycosides resistance.