AbstractDesorption electrospray ionization (DESI) mass spectrometry (MS) was used to differentiate seven bacteria species on the basis of their measured DESI‐mass spectral profile. Both gram‐positive and gram‐negative bacteria were tested and included Escherichia coli, Staphyloccocus aureus, Enterococcus sp., Bordetella bronchiseptica, Bacillus thuringiensis, Bacillus subtilis and Salmonella typhimurium. Distinct DESI‐mass spectra, in the mass range of 50–500 u, were obtained from whole bacteria in either positive or negative ion modes in less than 2 mins analysis time. Positive ion DESI‐mass spectral fingerprints were compared using principal components analysis (PCA) to investigate reproducibility for the intraday and the day‐to‐day measurements and the method selectivity to differentiate the bacteria studied. Detailed study of variances in the assay revealed that a large contribution to the DESI‐mass spectral fingerprint variation was the growth media preparation procedure. Specifically, experiments conducted with the growth media prepared using the same batch yielded highly reproducible DESI‐mass spectra, both in intraday and in day‐to‐day analyses (i.e. one batch of growth media used over a 3‐day period versus a new batch every day over the same 3‐day period). Conclusions are drawn from our findings in terms of strategies for rapid biodetection with DESI‐MS. Copyright © 2007 John Wiley & Sons, Ltd.