Investigation of bacterial food outbreaks by whole genome sequencing can rely on the inspection of the genetic relatedness between isolates through the application of single nucleotide polymorphism (SNP) thresholds. However, there is no consensus for Staphylococcus aureus in the context of food outbreaks. In this study, we propose a SNP cut-off by taking into account the mutation rate and the evolution time of this pathogen in food. Through in vitro microevolution, we determined the mutation rate of three S. aureus strains grown under mimicked food stressing conditions. From the mutation rate, we set a cut-off of 28 SNPs considering 30 days as evolution time based on the average shelf-life of foods contaminated by S. aureus and the timeline for identifying this pathogen in outbreaks. The SNP threshold was applied to retrospectively study ten staphylococcal food outbreaks to assess whether isolates from food and/or of human origin from the same outbreak were epidemiologically related. To interpret SNP distances, phylogenetic tree topologies and bootstraps were integrated and showed that isolates differing by up to 28 SNPs were monophyletic. Our suggested cut-off can be used in outbreak management to identify closely related S. aureus strains.