ABSTRACT: Thirty-nine carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates collected from a Chinese tertiary hospital were used in the characterization of the prevalence of 16S rRNA methylase genes. In total, 66.7% (26/39) of the CR-hvKP isolates were found to carry 16S rRNA methylase genes. The most frequently detected 16S rRNA methylase gene was armA (11/26, 42.3%), followed by rmtB (8/26, 30.8%), and coexistence of both armA and rmtB (7/26, 26.9%). All the clinical isolates were found to carry at least one carbapenemase gene, with blaKPC-2 (79.5%, 31/39), blaNDM-1 (10.3%, 4/39), and cocarrying blaKPC-2 and blaNDM-1 (10.3%, 4/39). A total of 89.7% (35/39) isolates carried extended-spectrum β-lactamase (ESBL) genes, including 61.5% (24/39) blaSHV-1, 71.8% (28/39) blaTEM-1, and 89.7% (35/39) blaCTX-M-14. All except four isolates (89.7%, 35/39) harbored quinolone resistance genes, with qnrS (82.1%, 32/39), aac(6')-Ib-cr (79.5%, 31/39), and qnrB (2.6%, 1/39). Twenty-six hvKP strains in this study were first reported to cocarry carbapenemase genes, ESBL genes, quinolone resistance genes, and 16S rRNA methylase genes simultaneously. Multilocus sequence typing (MLST) analysis assigned the 39 CR-hvKP isolates into 4 sequence types (STs), with ST11 encompassing 79.5% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 31 ST11 isolates. Cumulatively, 16S rRNA methylase genes are highly prevalent in CR-hvKP clinical isolates especially for ST11; it is, therefore, critical to continuously monitor the epidemiology of these 16S rRNA methylase-producing CR-hvKP while simultaneously minimizing potential risks from aminoglycoside-resistant CR-hvKP.