The present study investigates the suitability of direct bacterial profiling as a tool for the identification and subtyping of pathogenic Neisseria. The genus Neisseria includes two human pathogens , Neisseria meningitidis and Neisseria gonorrhoeae, as well as several nonpathogenic Neisseria species. Here , a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling protocol was optimized using a laboratory strain of E. coli DH5␣ to guarantee high quality and reproducible results. Subsequently , mass spectra for both laboratory and clinical strains of N. gonorrhoeae, N. meningitidis, and several nonpathogenic Neisseria species were collected. Significant interspecies differences but little intraspecies diversity were revealed by means of a visual inspection and bioinformatics examination using the MALDI BioTyper software. Cluster analysis successfully separated mass spectra collected from three groups that corresponded to N. gonorrhoeae , N. meningitidis , and nonpathogenic Neisseria isolates. Requiring only one bacterial colony for testing and using a fast and easy measuring protocol , this approach represents a powerful tool for the rapid identification of pathogenic Neisseria and can be adopted for other microorganisms.