A new fungal disease affecting wheat in Egypt, known as Stemphylium leaf spot, caused by Stemphylium vesicarium Wallr (Simmons) is reported. From all symptomatic wheat leaves, S. vesicarium was the most frequently isolated fungus (71.9%). The isolated pathogen was identified based on morphological characteristics together with molecular diagnosis. The Stemphylium isolate AUMC 15115 in this study was clustered at the same branch as Stemphylium mali CBS 122640, ex-type material (Synonym = Stemphylium vesicarium). At the Smart Agriculture Clinic Project, Sids Agricultural Research Station, Agricultural Research Centre, Beni Suef governorate, 12 cultivars of wheat were assessed in pot experiments for their resistance to Stemphylium leaf spot during the 2020 and 2021 growing seasons. Overall, distinct variations were observed in all examined cultivars in response to S. vesicarium infection. The Sakha 95 cultivar exhibited a minimal infection rate (disease incidence was 6.7, 3.3% and disease severity being, 0.7, 0.5%, respectively in the two growing seasons) and was classified as resistant. In contrast, Beni Suef 5 had the highest percent of infection (63.3, 66.7%), disease severity (38.5, 40.3%) and was classified as susceptible. In resistant cultivars, there was an increase in total phenol content, polyphenol oxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD) activities. Conversely, there was a reduction in electrolyte leakage percentage and hydrogen peroxide (H2O2) accumulation. However, the number of protein bands in resistant wheat cultivars exhibited a more significant increase than susceptible ones, particularly in the Sakha 95, which displayed the highest number of proteins.