AbstractApple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.