•Design of a multiplex PCR detecting genes rmtE- rmtH, npmA and subvariants.•94.5% (762/806) pan-aminoglycoside resistant Enterobacteriaceae had 16S RMTases.•In the UK 16S RMTases are associated with carbapenemases (93.4%, 712/762 isolates). The emergence of 16S rRNA methyltransferases (16S RMTases) worldwide is a growing concern due to their ability to confer high-level resistance (minimum inhibitory concentrations (MICs) >256 mg/L) to all clinically relevant aminoglycosides. As the occurrence of 16S RMTases in the United Kingdom has not been investigated to date, we screened 806 Enterobacteriaceae isolates displaying high-level aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) for 16S RMTases either by analysing whole-genome sequence (WGS) data (which were available for 449 isolates) or by polymerase chain reaction. A total of 94.5% (762/806) pan-aminoglycoside-resistant Enterobacteriaceae were positive for one or more 16S RMTase genes; armA was the most common (340, 44.6%) followed by rmtC (146, 19.2%), rmtF (137, 18.0%), rmtB (87, 11.4%) and various two-gene combinations (52, 6.8%). Most (93.4%; 712/762) 16S RMTase producers also carried acquired carbapenemase genes, with blaNDM the most common (592/712; 83.1%). Additionally, high-risk bacterial clones associated with blaNDM were identified in the subset of isolates with WGS data. These included Escherichia coli sequence types (STs) 405 (21.8%, 19/87), 167 (20.7%, 18/87) 410 (12.6%, 11/87) and K. pneumoniae STs 14 (35.6%, 112/315), 231 (15.6%, 49/315) and 147 (10.5%, 33/315). These accounted for 4.2% (15/358), 5.0% (18/358), 3.1% (11/358), 28.2% (101/358), 3.1% (11/358) and 7.0% (25/358) blaNDM-producing isolates, respectively. This study shows that 16S RMTases occur in the UK and Ireland and carbapenemases are particularly prevalent in 16S RMTase-producing Enterobacteriaceae. This association poses a risk to the treatment of multidrug-resistant Gram-negative infections in the clinical setting.