Objective To develop two rapid identification method of Cronobacter use polymerase chain reaction(PCR) and matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS), and to achieve high-throughput and low-cost screening of suspicious colonies of Cronobacter on chromogenic medium. Methods A pair of genus specific primers for Cronobacter were designed by internal transcribed spacer (its) gene, and 7 standard strains were set as reference strains to re-identify 214 strains which were preliminarily identified as Cronobacter collected in 2013. 84 isolates were selected randomly and identified by MALDI-TOF-MS and VITEK gram-negative bacteria identification card. Results The molecular weight of 7 reference strains were the same as expected, and PCR result of 214 isolates were positive. All 84 strains were identified as Cronobacter by both MALDI-TOF-MS and VITEK, and the coincidence rate was 100%. Conclusion PCR and MALDI-TOF-MS have the advantages of high-throughput, low-cost and short time consuming, which could achieve the preliminary screening of a large number of suspicious colonies, and could provide technical support for Cronobacter surveillance and control in China.