Abstract: Objective To investigate the virulence and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) in bloodstream infections. Methods CRKP strains initially isolated from bloodstream infections at Department of Laboratory Examination, the Affiliated Hospital of Xuzhou Medical University between June 2022 and March 2024 were collected. Antibiotic susceptibility testing was performed, and polymerase chain reaction (PCR) was used to detect carbapenemase genes (bla_KPC and bla_NDM). Clinical data was collected. Pulsed-field gel electrophoresis (PFGE) was conducted to assess the homology of strains carrying the bla_KPC gene. Meanwhile, the virulence of the strains was evaluated by in vitro serum killing assay and Galleria mellonella infection model. Results A total of 22 CRKP strains were collected, all of which were resistant to penicillins, cephalosporins, and carbapenems, with some showing resistance to polymyxin and tigecycline. All patients had invasive procedures, and most had underlying diseases. Homology analysis of the strains revealed seven clonal types. The virulence of strain KP21 was higher than that of the high-virulence control strain. Conclusions All CRKP strains exhibit high resistance to multiple antibiotics and include highly virulent strains. Monitoring of these highly pathogenic CRKP strains should be strengthened to control their spread within hospitals.