Pseudomonas syringae pv. syringae causes extensive yield losses in wine-grape production in some Australian cool-climate vineyards. Putative P. syringae pv. syringae isolates from infected grapevines within a range of vineyards were genotyped using RNA polymerase ÃŽÂ²-subunit (rpoB) and multilocus sequence typing (MLST) using primers for glyceraldehyde-3-phosphate dehydrogenase (gapA), citrate synthase (gltA), DNA gyrase B (gyrB), and Ã�' factor 70 (rpoD). The isolates were also evaluated for pathogenicity by inoculation of detached grapevine leaves. The isolates were grouped by MLST data into two well-supported clades, each containing a mixture of pathogenic and nonpathogenic grapevine isolates, indicating that P. syringae pv. syringae in Australian vineyards is genetically diverse. Each clade also contained P. syringae pv. syringae from nongrape hosts pathogenic to grapevine, demonstrating a lack of host specificity and possible potential for cross-infection of grape and other horticultural crops. Furthermore, the isolation of pathogenic P. syringae pv. syringae isolates from grapevine sucker shoots suggests that sucker shoots may allow overwintering of the pathogen. The vineyard quarantine status of P. syringae pv. syringae may need to be reconsidered, due to its easy dispersal through pruning equipment.