Using morphological and molecular methods, this research sought to distinguish, classify, and characterize the pathogen associated with pokkah boeng disease (knife-cut symptoms). During the 2021 growing season, the sugarcane breeding program at El-Mattana Agricultural Research Station, Luxor Governorate, Egypt, the knife-cut symptom was observed on stalks of sugarcane (variety G.2009-99). The causal pathogen was isolated from samples taken from infected stalks with knife-cut lesions. Pathogenicity studies affirmed that the pathogen isolate was pathogenic on cuttings of the commercial sugarcane variety G. T. 54-9. After 7 days from inoculation, disease signs were detected in cane stalks when opened longitudinally. Stalks of sugarcane in control treatments showed no signs of disease. The pathogen was re-isolated and determined to be similar to the causal fungus that triggered knife-cut symptoms in a normal infection in the field. Morphological characteristics of growth rates, colony features, and shapes of macroconidia, microconidia, conidiogenous cells, and chlamydospores were used to identify the isolate. The pathogen internal transcribed spacer (ITS) was sequenced using ITS1 and ITS4 primers to classify and differentiate it. After microscopic morphology and gene sequencing, the fungus isolate was identified as Fusarium verticillioides. The pathogen pure culture was preserved in the Assiut University Mycological Centre as AUMC 14795, and the ITS sequence was uploaded to GenBank as MW692089. More research is required to assess the prevalence of the pathogen, the resistance of various sugarcane varieties, and an effective method for managing the pathogen.