Accurate detection of Salmonella in food and Listeria spp. in processing environments is crucial to food safety initiatives, thus reliable Salmonella and Listeria spp. detection methods are an essential component of a facility’s food safety plan. This work highlights the critical role strain and species selection play in Salmonella and Listeria spp. method evaluations. For the Salmonella work, three PCR-based Salmonella detection methods were evaluated for their ability to detect Salmonella from a low-moisture food inoculated with difficult-to-detect strains. Incorporated into this study was an AOAC-validated alternative enrichment offering both cost and time savings compared to the reference method procedure. This study identified a need to optimize the alternative enrichment procedure as a significant risk of false negatives and/or PCR-inhibition was detected; this same risk was not reported in the AOAC study. The Listeria work focused on recently described species via two different studies, one evaluating three Listeria reference methods (FDA, USDA, ISO) for their ability to grow all species, and another describing six novel Listeria species. Both studies generated several key findings that were not previously reported. The reference method study identified that several recently described sensu lato species may lead to Listeria spp. false negatives. Of the various mechanisms that could lead to a false negative (e.g., limited growth), one interesting finding was the potential for several recently described sensu lato species to outgrow L. monocytogenes indicator or index organisms (the sensu stricto species L. seeligeri or L. ivanovii); this potential was observed with the ISO and USDA enrichment procedures, but not with the FDA. The novel Listeria species work describes four novel sensu stricto and two novel sensu lato species. Notably, this worked revised the phenotypic expectations for Listeria sensu stricto species (e.g., motility is not no longer universal). Additionally, if a novel Listeria sensu stricto species is present and detected it will likely be misidentified as another sensu stricto species, including potentially L. monocytogenes or L. ivanovii.