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Optical detection of foodborne bacteria such as Salmonella classifies bacteria by analysing spectral data, and has potential for rapid detection. In this experiment hyperspectral microscopy is explored as a means for classifying five Salmonella serotypes. Initially, the microscope collects 89 spectral measurements between 450 and 800 nm. Here, the objective was to develop correct classification of five serotypes with optimal spectral bands selected through multivariate data analysis (MVDA), thus reducing the data processing and storage requirement necessary for practical application in the food industry. An upright digital microscope is equipped with an acousto-optical tuneable filter, electron multiplying charge-coupled device, and metal halide lighting source. Images for each of the five serotypes were collected, and informative bands were identified through a principal component analysis, for four abbreviated spectral ranges containing 3, 7, 12 and 20 spectral bands. The experiment was repeated with an independent repetition and images were collected at each of the reduced band sets, identified by the first repetition. A support vector machine (SVM) was used to classify serotypes. Results showed that with the first repetition, classification accuracy decreased from 99.5% (89 bands) to 84.5% (3 bands), whereas the second repetition showed classification accuracies of 100%, possibly due to a reduction in spectral noise. The support vector machine regression (SVMR) was applied with cross-validation, and had R(2) calibration and validation values >0.922. Although classification accuracies through SVM classification showed that as little as 3 bands were able to classify 100% of the samples, the SVMR shows that the smallest root-mean squared-error values were 0.001 and 0.002 for 20 and 12 bands, respectively, suggesting that the 12 band range collected between 586 and 630 nm is optimal for classifying bacterial serotypes, with only the informative HMI bands selected., (Published [2015]. This article is a U.S. Government work and is in the public domain in the USA.)