BACKGROUND: Development of polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a severe challenge to public health. Here we aimed to perform a retrospective study of prevalence and molecular characteristics of polymyxin-resistant CRKP strains. METHODS: 4455 clinical CRKP strains from 18 provinces in China during 2000 to 2023 were collected. Polymyxin-resistant CRKP strains were subjected to antimicrobial susceptibility testing, whole genome sequencing and bioinformatic analysis. Molecular mechanisms underlying the polymyxin resistance in CRKP were analyzed. RESULTS: Here we show that polymyxin-resistant CRKP emerge initially in 2014, prevalence of such strains then increase steadily over the years, reaching a rate of 9.86% in 2023. In total, 112 polymyxin-resistant CRKP isolates are identified. Antimicrobial susceptibility tests show that all polymyxin-resistant CRKP are resistant to commonly used antibiotics, yet most isolates remain susceptible only to ceftazidime-avibactam and tigecycline. Predominant polymyxin resistance mechanism in CRKP is mutations in mgrB (59/112), which commonly involves disruption of mgrB by insertion of elements such as ISKpn26 (20/59), IS903B (14/59), and ISKpn14 (9/59). Phylogenetic analysis reveals frequent clonal dissemination of polymyxin-resistant CRKP within the same hospital and even among different hospitals in neighboring provinces. pLVPK-like virulence plasmids are detected in 46 isolates, such strains are therefore categorized as polymyxin and carbapenem-resistant hypervirulent K. pneumoniae which may cause infections with high mortality. CONCLUSIONS: Our results highlight frequent clonal transmission of polymyxin-resistant CRKP within hospitals. Continuous surveillance of polymyxin resistance among CRKP should be implemented to prevent further dissemination of such strains in clinical settings in China.