Dickeya, a genus of important and high consequence bacterial plant pathogens, is listed among the quarantine pathogens of the European Union and is associated with potato disease outbreaks and economic losses worldwide. Early, accurate, sensitive and reliable detection of the pathogen is needed to prevent further dissemination and establishment of disease. A genome-informed multiplex TaqMan qPCR labeled with different fluorophores was developed for accurate and sensitive detection of Dickeya pathogens. A whole genome comparative genomics approach was used to identify unique and conserved regions: alcohol dehydrogenase in D. dianthicola strains and mglA/mglC for the genus Dickeya. The developed multiplex TaqMan qPCR was validated using an inclusivity panel to confirm broad range detection capability and an exclusivity panel to exclude cross-reactivity with strains of closely related genera and species. The developed assay was validated with plant samples naturally and artificially infected with Dickeya and/or D. dianthicola. Both sensitivity and spiked sensitivity assays showed the limit of detection down to 10 fg/reaction. No false positives or false negatives were detected. This assay will serve as a practical tool for screening propagative material, monitoring the presence and distribution of pathogens, and quantification of target pathogens in a breeding program. It also has applications in routine diagnostics, biosecurity, and microbial forensics.