Objective To screen specific mass peaks(SMP) of carbapenem-resistant Klebsiella pneumoniae (CRKP) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and perform rapid homology analysis on CRKP based on SMP. Methods 76 strains of CRKP with multilocus sequence typing (MLST) were selected from a microbiology laboratory of a hospital, 52 strains were for establishment of method and 24 strains were for validation of method. Five different criteria for selecting SMP of CRKP were set, SMP of different ST-type were screened according to different criteria, CRKP was typed based on screened SMP, criterion with the highest coincidence rate of MLST results was determined as the best criterion. Twenty-four CRKP strains were typed according to the specific peaks screened through best criteria, accuracy of SMP typing method was verified, and was compared with principal component analysis (PCA) and main spectra profile(MSP) cluster analysis in mass spectrometer software. Results According to standard 2 (1signal-to-noise ratio[S/N] ≥ 4, 2S/N ratio ≥ 1.5, 3coefficient of variability[CV ≤ 40%]), 45 SMP were selected from 52 strains of CRKP strains, 29 strains of CRKP were typed by SMP, with the highest coincidence rate (82.8%) with MLST, which was determined as the best criterion. Another 24 CRKP strains were typed according to SMP screened based on this criterion, and the coincidence rate with MLST was 83.3%. The coincidence rate between PCA cluster analysis and MLST was only 66.7%, consistency between MSP clustering analysis and MLST was poor, and it didn't conform to the grouping trend of ST typing. Conclusion MALDI-TOF MS can select SMP of CRKP of different ST, which can be used for rapid homology analysis on CRKP, provide basis for surveillance and control of outbreak of healthcare-associated infection.