Cronobacter spp. are Gram-negative opportunistic food-borne pathogens and are known as rare but important causes of life-threatening neonatal infections. Rapid and reliable identification of Cronobacter species and their differentiation from phenotypically similar, nonpathogenic Enterobacter turicensis, Enterobacter helveticus, and Enterobacter pulveris have become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid genus and species identification of the six Cronobacter species recognized so far. To this end, we developed a reference MS database library that includes 54 Cronobacter target strains as well as 17 nontarget strains. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2,000 to 30,000 Da). Genus- and species-specific biomarker protein mass patterns were determined. The defined biomarker mass patterns (Spectral Archive and Microbial Identification System [SARAMIS] SuperSpectrum) were validated using 36 strains from various Cronobacter species as well as eight nontarget strains. For all strains the mass spectrometry-based identification scheme yielded identical results as with a PCR-based identification system. All strains were correctly identified, and no nontarget strain was misidentified as Cronobacter. Our study demonstrates that MALDI-TOF MS is a reliable and powerful tool for the rapid identification of Cronobacter strains to the genus and species level.