This study investigated the virulence, pathogenicity, and population structure of Neonectria ditissima, the causative agent of European canker (EC) in apple. Twelve isolates were phenotyped for virulence and pathogenicity in a glasshouse assay using one-year-old 'Katja' apple trees. Significant differences in virulence were observed among isolates, with area under disease progression curve (AUDPC) values ranging from 58.66 to 4347.79. Disease incidence varied considerably among isolates and was strongly correlated with virulence (r = 0.85, p < 0.001). The estimated broad-sense heritability for virulence was remarkably high (H² = 0.98), indicating that variation in AUDPC was predominantly attributable to genetic differences between isolates. Population structure was analyzed using simple sequence repeat (SSR) markers for 25 isolates and whole genome sequencing (WGS) data for 38 isolates. Principal coordinate analysis of SSR data suggested potential clustering patterns, though not clearly correlated with geographic origin or virulence. Principal component analysis of WGS data revealed clustering patterns related to both host of origin and geographic distribution. Isolates from Fagus hosts grouped closely despite diverse geographic origins, while isolates from different European regions showed distinct clustering patterns. Additionally, ITS sequencing of 46 isolates, including related Neonectria species, confirmed the species identity of the studied isolates and demonstrated the ability of ITS sequences to differentiate most Neonectria species except N. ditissima and N. major. This research provides valuable insights into the genetic diversity and virulence characteristics of N. ditissima, which will inform resistance breeding strategies for apple and improve understanding of host-pathogen interactions in European canker disease
