Bacterial populations are highly diverse on horticultural crops. Many species are comprised of both pathogenic and non-pathogenic variants, which can lead to limitations in existing taxonomic-based molecular diagnostics. This study used the kiwifruit-Pseudomonas pathosystem as a model to develop alternative molecular diagnostics that might assist in rapidly defining risks of bacteria at the border. Initially, a collection of pseudomonads from kiwifruit were tested for virulence. Loci that differentiated pathogenic pseudomonads from non-pathogenic strains were then identified using comparative genomics. Finally, PCR was optimized to target these loci, providing a more generic risk assessment of unknown strains. Our new knowledge of pathogen-associated loci was subsequently applied to define the biosecurity risk from Pectobacterium carotovorum subsp. actinidiae (Pca), an unrelated enterobacterium that purportedly causes summer cankers in kiwifruit orchards throughout South Korea. Future work will also examine whether pathogen-associated loci in other important genera may aid biosecurity decision making, especially in post entry quarantine, surveillance and incursion response.