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