Pectobacterium, a pectinolytic phytobacterium, is listed among the top 10 most destructive plant pathogens. The newly defined species P. parmentieri, which lacks a type III secretion system (T3SS), has emerged as a particularly aggressive pathogen by eliciting soft rot and blackleg diseases that have impacted the potato productivity and jeopardized food security. The purpose of this research was to develop a robust and highly accurate multiplex TaqMan qPCR to specifically detect all known Pectobacterium species and the recently characterized P. parmentieri. Comparative genomic approaches were used to determine conserved signature genomic regions for the genus and species. A triplex TaqMan qPCR was developed to target unique gene regions of Pectobacterium and P. parmentieri, and a universal internal control (UIC) was incorporated to enhance the reliability of the assay. The assay was validated using diverse strains, meticulously selected for exclusivity and inclusivity panels. No false positives or false negatives were detected during the assay validation. The detection limit of qPCR is down to 10- and 100-fg per reaction for Pectobacterium and P. parmentieri, respectively. The assay developed in this study will contribute to tracking the geographic distribution and dissemination of Pectobacterium, and will be an excellent tool for detecting and quantifying the target species especially in seed certification programs, microbial forensics and routine diagnostics.