During the period 2006–2011, Pseudomonas syringae pv. syringae caused a bacterial inflorescence rot (BIR) epidemic in an Australian cool climate viticultural region. Molecular multilocus sequence typing of ‘housekeeping’ genes (MLST), biochemical testing and analysis of molecular variance (AMOVA) were used to characterize the genotypes and phenotypes of P. syringae pv. syringae grapevine isolates. Comparison of the MLST data with exemplars of phylogroups available at PAMDB demonstrated that the BIR isolates formed a new clade within P. syringae pv. syringae phylogroup 2 (PG02): putatively designated PG02f. Analysis of the MLST and phenotypic data by AMOVA demonstrated some genetic differences between the BIR isolates and the general vineyard P. syringae pv. syringae population. Isolates positive for syringopeptin, syringomycin and tyrosinase, tobacco leaf hypersensitivity reaction (HR), ampicillin resistance and grapevine leaf pathogenicity were genetically distinct from those negative for these factors. This study has shown that, generally, the core genome of P. syringae pv. syringae is only weakly associated with the virulence associated traits. As the new phylogroup PG02f consists of the epidemic BIR isolates and nonpathogenic grapevine isolates, these genetically similar isolates differ greatly in pathogenicity and most of the other tested phenotypic traits. However, within the PG02f group, tobacco leaf HR and presence of syle (the gene for phytotoxin syringolin A) are associated with the BIR and bacterial leaf spot (BLS) isolates, and negative for the nonpathogens, indicating that these two virulence factors may be associated with vineyard pathogenicity within the new Australian phylogroup.