Objective To analyze the molecular epidemiology and colistin-resistant genes of carbapenem-resistant Klebsiella pneumoniae (CRKP) by whole-genome sequencing, and to provide reference for clinical diagnosis and treatment. Methods 57 CRKP strains isolated from clinical specimens of hospitalized patients in a tertiary general first-class hospital in Anhui Province from 2021 to 2023 were collected and antimicrobial susceptibility testing was performed. Multilocus sequence typing, capsule serotype, resistance genes, and virulence genes of CRKP strains were analyzed by whole-genome sequencing technique, and single nucleotide polymorphism analysis was conducted on sequences of all strains. Colistin resistance-related genes were amplified by polymerase chain reaction (PCR). Results 57 CRKP strains exhibited resistance to 14 antimicrobial agents, with the exception of tigecycline. The sequencing results showed that 93.0% (53/57) of CRKP carried bla_KPC-2, and the ST11 type CRKP strain had the highest detection rate (51/57, 89.5%). Single nucleotide polymorphism clustering analysis showed that the 57 CRKP strains were divided into 11 clone groups, of which 4 clone groups were all ST11-KL64 type CRKP. 40 (70.2%) CRKP strains carried multiple virulence genes. Five strains of CRKP were colistin-resistant strains, the resistance mechanism involved the insertion of ISKpn26 element at site 70 of the mgrB gene. Conclusion The CRKP strain is primarily characterized by the production of KPC-2 ST11-KL64, with disseminated transmission in intensive care unit. The insertion of ISKpn26 element leading to mgrB gene mutation is related to resistance of CRKP to colistin in this region.