The objective of this study was to develop a method for the rapid detection and differentiation of Salmonella serovars using immunomagnetic separation (IMS) combined with Fourier-Transform Infrared (FT-IR) microspectroscopy and multivariate analysis. Selected Salmonella serovars, implicated in foodborne disease outbreaks were grown in tryptic soy broth for 8, 18 and 24 h at 35 °C. Anti-Salmonella magnetic beads (Dynabeads®) were added to the culture to specifically isolate and concentrate Salmonella. Bacteria-bead complexes were aseptically applied onto a hydrophobic grid membrane, dried under vacuum and analyzed by attenuated total reflectance using a FT-IR microspectroscopy. Spectral data were used to create soft independent modeling of class analogy models for Salmonella differentiation. Application of IR microspectrometry provided sensitivity and resolution of unique chemical fingerprints to allow detection and differentiation of Salmonella strains due to differences in lipopolysaccharides (985 cm-1) of the cell envelope. Salmonella cells bound to immunomagnetic beads had distinctive and reproducible infrared spectra and allowed characterization of particular bacterial structures but interference signal from the beads in the fingerprint region prevented accurate differentiation at the serovar level. Results indicated that binding of the beads to Salmonella differed for various serovars. © 2010 The Royal Society of Chemistry.