During the annual assessment of the National Wheat Program, Gemmeiza Agricultural Research Station, Gharbia governorate, March 2021, brown spots were observed on the spikes of A-ESWST and SAWYT experiments. These experiments were originated from International Maize and Wheat Improvement Center (CIMMYT) to assess the yield and wheat diseases under Egyptian environmental conditions. Disease severity on the infected spikes was up to 30%. At the end of the season, all the infected spikelets turned to dark brown color. The pathogen was identified morphologically as Stemphylium vesicarium in the Mycology Dept., Plant Pathology Institute, ARC, Giza and in the Regional Center of Mycology and Biotechnology Al-Azhar University. The culture includes conidiophores, 3.0 μm and conidia in short chains and conidia are ovoid and obclavate 32.6×9.6 μm, with 3-8 transverse and several longitudinal septa. For molecular identification of Stemphylium isolate, total genomic DNA was extracted and amplified using the primers Internal Transcribed spacer (ITS1 and ITS4) then sequenced. The sequenced DNA was aligned by BLAST and searched for homology in the GenBank nucleotide database. Molecular identification confirmed that the isolate is Stemphylium vesicarium with 100 % homology with other Stemphylium stains and various accession numbers in NCBI data Bank. The sequence was submitted to NCBI and has accession number OM722056. To fulfill Koch's postulates, wheat plants in 2022 growing season at flowering stage were sprayed with Stemphylium vesicarium suspension with concentration 2.5×10⁴ conidia ml⁻¹. The same symptoms appeared on wheat spikes with 100% disease severity and S. vesicarium was reisolated and have the same microscopic characters as initial culture.