Objective：Matrix-assisted laser desorption/ionization-time of flight mass spectrometry（MALDI-TOF MS）was performed to detect the homology of carbapenem-resistant Klebsiella pneumoniae（CRKp）. Compared with pulsed field gel electrophoresis（PFGE）and multilocus sequence typing（MLST），the application value of mass spectrometry in daily work was discussed. Methods：Ten strains of Klebsiella pneumoniae isolated from patients in our hospital from October 2017 to December 2017 and collected from ICU environment were collected. Eight resistant genes（blaTEM，blaSHV，blaCTX-M，blaKPC，blaIMP，blaNDM，blaVIM，and blaOXA）were detected. The homology of these strains was analyzed by PFGE，MLST and mass spectrometry（MALDI-TOF MS），and the results were compared. Results：It was confirmed that 10 strains of CRKp were all of the same cloned strains by PFGE，the ST-type of ST11. MALDI-TOF MS could be distinguished from different strains of Klebsiella pneumoniae by cluster analysis and principal component analysis. The homology of the same type of Klebsiella pneumoniae was about 80%. Conclusion： MALDI-TOF MS can be used in the detection of the homology of Klebsiella pneumoniae in nosocomial local infection，which is consistent with the results of PFGE and MLST. But it is necessary to establish the standard judgment standard as soon as possible.