Identification of bacteria by mass spectrometry offers the potential of a high-throughput non-targeted method to determine the presence of Salmonella. While MALDI-TOF mass spectrometry can identify Salmonella at the genus and species level, few studies have reported subtyping beyond the species level due to the diversity and complexity of Salmonella that includes more than 2600 serovars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches enable profiling of a greater number of proteins over a larger dynamic range and offer the potential to detect small differences between closely related isolates. We evaluate the discriminatory power of bottom-up LC-MS/MS with a collection of nineteen isolates that differ at the genus, species, subspecies, or strain level. Isolates were classified by matching the sequence of identified peptides to reference proteomes translated from genomes with known taxonomic ranks. The degree of proteomic similarity between the tested isolates and reference strains correlated with how closely they were related. All tested Salmonella isolates were easily distinguished from their close relatives, E. coli and Shigella, and readily grouped by species and subspecies. Additionally, each Salmonella isolate most closely matched to its correct serovar. This approach presents a simple and effective proteomic approach to identification of Salmonella genus, species, and subspecies.