Polymyxin was recently approved for the clinical treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections in China. The aim of this study is to explore the prevalence and molecular mechanisms of polymyxin resistant CRE (PR-CRE) prior to the clinical application of polymyxin, and to evaluate the potential of emerging polymyxin resistance in China. 504 unique CRE isolates were collected from six tertiary care hospitals in China between October 2016 and September 2017. All isolates were subjected to antimicrobial susceptibility testing, while clinical, demographic, antimicrobial exposure, and infection data were collected from medical charts. PCR detection, Sanger sequencing and real-time fluorescence quantitative PCR (RT-qPCR) were used to investigate the molecular mechanism of the polymyxin resistance. A total 19 (3.8%) polymyxin resistant isolates were identified, including Klebsiella pneumoniae, Escherichia coli, K. aerogenes and Enterobacter cloacae. Genetic analysis in K. pneumoniae strains identified insertion sequence (IS) (n=3), stop codon (n=1) and gene deletion (n=2) in mgrB, and pmrB missense mutation (T157P) (n=1). Two E. coli contain mcr-1 and an E. cloacae strain harbors a frameshift in mgrB. Further transcriptional analysis showed that pmrA, pmrB, pmrC and pmrK gene were significantly upregulated in polymyxin resistant isolates. Despite without polymyxin exposure, ∼3.8% CRE isolates were resistant to polymyxin in China. Both chromosome and plasmid-encoding mechanisms were identified. Our study suggested that clinical practice should be alert to the pre-existed polymyxin resistance among CRE isolates to avoid the further dissemination of polymyxin resistance. Copyright © 2020. Published by Elsevier Ltd.