Background <p>Bacterial canker, caused primarily by <i>Pseudomonas syringae</i> pv. <i>syringae</i> (<i>Pss</i>), is a major constraint to sweet cherry (<i>Prunus avium</i> L.) production worldwide. The development of resistant cultivars is hindered by the perennial nature of the crop, the genetic diversity of the pathogen, and the lack of reliable high-throughput screening methods that correlate with field performance. This study aimed to develop and validate a rapid, reproducible assay for assessing sweet cherry susceptibility to <i>Pss</i> that accurately reflects field observations and accounts for pathogen diversity. A three-year field survey (2017–2019) across 222 orchards in Chile’s O’Higgins Region established the susceptibility spectrum of major cultivars, identifying 'Santina' (low susceptibility), 'Lapins' (moderate susceptibility), and 'Bing' (high susceptibility) as reference cultivars. A collection of 27 Chilean <i>Pss</i> strains was characterized for virulence. Leaf disc assays (LDA) were evaluated under different light regimes and compared with field twig inoculations and orchard data. Bacterial growth was quantified using a <i>Pss</i>-specific qPCR assay.</p> Results <p>Light conditions critically influenced assay outcomes. Under continuous darkness, the susceptibility pattern inverted completely, with 'Bing' appearing less susceptible than 'Santina'. Only under a 16-h photoperiod did the LDA reproduce the field-observed susceptibility gradient ('Santina' &lt; 'Lapins' &lt; 'Bing'). The LDA results were strongly and positively correlated with the mean field severity index. For strain A1M3, Pearson’s correlation coefficient was 0.79 (95% CI: 0.32–0.95, <i>p</i> = 0.006); for strain A1M250, it was 0.81 (95% CI: 0.38–0.95, <i>p</i> = 0.004). The corresponding Spearman’s rank correlations were even higher (<i>ρ</i> = 0.83 and 0.85, respectively), indicating that the rank order of cultivars based on either lab test reliably predicts their rank order in the field. In contrast, the twig assay showed no significant correlation with field severity (<i>r</i> = 0.11, <i>p</i> = 0.77; <i>ρ</i> = 0.12), suggesting that mature twig tissue or specific assay conditions did not adequately capture the genetic susceptibility expressed in whole trees under natural infection.</p> <p>The Chilean <i>Pss</i> population comprised four distinct virulence groups, including a hypervirulent strain (A1M200) capable of causing severe damage (&gt; 64%) even in the resistant cultivar 'Santina'. Strain-specificity analysis revealed significant cultivar–strain interactions, with several strains showing adaptation to 'Bing'. Quantitative qPCR demonstrated that reduced susceptibility in 'Santina' was associated with approximately five-fold lower bacterial loads compared to 'Bing', suggesting that resistance operates through growth restriction rather than pathogen elimination.</p> Conclusions <p>The validated leaf disc assay conducted under a 16-h photoperiod provides a rapid, cost-effective tool for early-stage selection in sweet cherry breeding programs. By capturing the quantitative nature of resistance and accounting for pathogen diversity, this assay can accelerate the development of cultivars with durable resistance to bacterial canker, contributing to the long-term sustainability of the cherry industry.</p>