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Subtyping of bacterial isolates of the same genus and species is an important tool in epidemiological investigations. A number of phenotypic and genotypic subtyping methods are available; however, most of these methods are labor-intensive and time-consuming and require considerable operator skill and a wealth of reagents. Matrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF), an alternative to conventional subtyping methods, offers a rapid, reproducible method for bacterial identification with a high sensitivity and specificity and at minimal cost. The purpose of this study was to determine the feasibility of using MALDI-TOF to differentiate between six Salmonella serovars recovered from experimental microcosms inoculated with known strains of Salmonella. Following the establishment of a MALDI-TOF reference library for this project, the identity of 843 Salmonella isolates recovered from these microcosms was assessed using both MALDI-TOF and conventional methods (serotyping/PCR). All 843 isolates were identified as being Salmonella species. Overall, 803/843 (95%) of these isolates were identified similarly using the two different methods. Positive percent agreement at the serovar level ranged from 79 to 100%, and negative percent agreement for all serovars was greater than 98%. Cohen’s kappa ranged from 0.85 to 0.98 for the different serovars. This study demonstrates that MALDI-TOF is a viable alternative for the rapid identification and differentiation of Salmonella serovars.