ABSTRACT Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes ( p ilW and p ilX ) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444- to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 × 10 ² CFU g ⁻¹ or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen.