Aims: The development and evaluation of a sensitive and specific TaqMan (R) real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results: A TaqMan (R)-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan (R) PCR assay detected 1 pg of purified DNA and 10(4)P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92.0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan (R) real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.