Objective VITEK matrix-assisted laster desorption ionization-time of flight mass spectrometry (VITEK MALDI-TOF MS,hereinafter referred to as VITEK MS). To study the impact of different sample preparation procedures for Cronobacter sakazakii identification and its traceability. And two data acquisition softwares of VITEK MS and MALDI 7090 were used to compare the differences of mass spectrograms between them. Methods Twenty two strains, including 18 isolated Cronobacter sakazakii strains, two Cronobacter sakazakii reference strains, one Enterobacter cloacae reference strain and one Citrobacter freundii reference strain were prepared respectively by three different preparation procedures, including direct transfer method, formic acid extraction method and formic acid-acetonitrile extraction method, and analyzed by MS. A detailed analysis of mass spectrograms was obtained from the software of VITEK MS. The result were compared with biochemical result. Then, the impact of sample preparation method on the identification result was evaluated by researching the consistency of result derived from three different preparation method, The traceability of 20 Cronobacter sakazakii strains was also investigated by the cluster analysis of VITEK MS result. The same strains were analyzed by MALDI 7090, and the mass spectrograms were compared with those by VITEK MS. Results The 20 strains were identified by VITEK MS as Cronobacter sakazakii, which were in consistent with biochemical result and could be distinguished from Enterobacter cloacae and Citrobacter freundii. There was no significant difference in the result confidence among the three different preparation procedures(P>0.05), but the number of mass peaks and their relative intensities showed remarkable difference. The formic acid extraction method achieved spectra consisting of >15 peaks and mass ranges of 2 000-15 000 m/z, of which relative intensities were >5 mV. At similarity level of 80%, the 20 strains prepared by the formic acid extraction method were classified into 5 clusters by the cluster analysis of VITEK MS result, through which the transmission route of Cronobacter sakazakii could be identified. There was no obvious difference in the peak positions and relative intensity between VITEK MS and MALDI 7090. Conclusion MALDI-TOF MS was considered as a reliable technique in the identification of Cronobacter sakazakii. The formic acid extraction method was more suitable for the identification of Cronobacter sakazakii by MALDI-TOF MS, which will be a useful technique for Cronobacter sakazakii trace. There was no obvious difference in the mass spectrograms between VITEK MS and MALDI 7090.