Aims: The purpose of this study was to investigate the role of H2O2 and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H2O2-specific scopoletin fluorometric assay and the CAT-dependent H2O2 consumption assays, it was found that the production rate of intra/extracellular H2O2 and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H2O2 is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H2O2 is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H2O2 levels.