Introduction:Colistin-resistant carbapenem-resistant Klebsiella pneumoniae (ColR-CRKP) is a threat to public health safety. The main cause of polymyxin resistance is a change in the negative charge and L-Ara-N and PEtN molecules on the cell membrane, which leads to a decrease in the affinity of polymyxin for the strain. Methodology: In this study, (ColR-CRKP) isolates and clinical data were collected from a tertiary teaching hospital in China .Testing of antimicrobial susceptibility explained on the European Committee on Antimicrobial Susceptibility Testing criteria (EUCAST), Mutations and expression of drug-resistant genes were detected by PCR and RT-PCR. Genotyping of isolates analysised by MLST and PFGE. Results:Fourteen ColR-CRKP strains were collected and all isolates were resistant to colistin and most other clinical antibiotics (except tigecycline and cotrimoxazole), and were divided into 6 different PFGE clusters, and most strains clustered in B,C,D groups. Base mutations in the mgrB gene that can lead to changes in amino acid sequence were found in all 14 strains. Missense mutations a55→t translated into S32C were found in 85.7% isolates (n=12; KP1-9, KP11-12 and KP14). The expression level of mgrB gene was decreased in all ColR-CRKP strains. Conclusions:Third-generation cephalosporins and enzyme inhibitors, as well as carbapenems, may be causative for the mutation. The new challenge for the treatment of ColR-CRKP requires broader attention.Our study showed that mutations in the mgrB gene can lead to the development of polymyxin resistance in in ST11 and ST15-KPC-2-producing K. pneumoniae in this region.