Objective To explore the homology of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from patients and environment in an intensive care unit (ICU) of a hospital, and provide theoretical basis for the prevention and control of healthcare-associated infection(HAI). Methods CRKP strains continuously isolated from patients and environment in ICU in a tertiary first-class hospital between September and December 2017 were collected, drug resistance phenotype and carbapenemase genes were detected, molecular homology analysis was performed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results A total of 10 strains of CRKP were collected, 9 of which were isolated from clinical infection specimens of 5 patients, and 1 was isolated from the panel of the barotherapy instrument. 10 strains all carried KPC gene; MLST types were all ST11; there were 5 types of PFGE bands, 5 strains had identical bands, which were epidemic strains, 1 strain was only 1 band different from epidemic strain, the other 4 strains had similar band types, but they were significantly different from that of the epidemic strain. Drug resistance phenotype, PFGE type and ST type of 1 CRKP strain isolated from panel of barotherapy instrument was identical with 4 CRKP strains from patients, and was considered as HAI due to instrument sharing. PFGE band type of CRKP strains isolated from the abdominal drainage fluid and blood specimen of the same patient were exactly the same, which was speculated that CRKP might be infected bloodstream through the abdominal cavity. Conclusion PFGE and MLST are of great significance to clarify bacteria transmission route of HAI, individual bacterial infection route and genetic variation, which is conductive to cutting off infection route and strongly guiding the control of multidrug-resistant organism transmission.