| Summary: | <i>Enterobacteriaceae</i> is one of the most important bacterial groups within the Proteobacteria phylum. This bacterial group includes pathogens, commensal and beneficial populations. Numerous <i>16S rRNA</i> gene PCR-based assays have been designed to analyze <i>Enterobacteriaceae</i> diversity and relative abundance, and, to the best of our knowledge, 16 primer pairs have been validated, published and used since 2003. Nonetheless, a comprehensive performance analysis of these primer sets has not yet been carried out. This information is of particular importance due to the recent taxonomic restructuration of <i>Enterobacteriaceae</i> into seven bacterial families. To overcome this lack of information, the identified collection of primer pairs (n = 16) was subjected to primer performance analysis using multiple bioinformatics tools. Herein it was revealed that, based on specificity and coverage of the <i>16S rRNA</i> gene, these 16 primer sets could be divided into different categories: <i>Enterobacterales-</i>, multi-family-, multi-genus- and <i>Enterobacteriaceae</i>-specific primers. These results highlight the impact of taxonomy changes on performance of molecular assays and data interpretation. Moreover, they underline the urgent need to revise and update the molecular tools used for molecular microbial analyses.
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