Validation data is presented for a conventional PCR test that specifically detects the quarantine pathogen Pantoea stewartii subsp. stewartii in maize leaf and seed samples and does not cross‐react with the non‐pathogenic P. stewartii subsp. indologenes. The PCR tests currently recommended by the EPPO Diagnostic Protocol PM 7/60 (2) for initial screening bear the risk of false positives as they detect both subspecies of P. stewartii. The test presented here has high analytical sensitivity (102 cells mL–1 in leaf extracts, at least 103 cells mL−1 in seed extracts), high repeatability and high reproducibility and performed well on the two matrices tested: maize seed and leaf extract. Its improved analytical specificity and analytical sensitivity compared to the currently recommended tests lead the authors to suggest that this test should be included as a first screening test in the diagnostic protocol for P. stewartii subsp. stewartii when it is next revised.