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Abstract Background The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker. Results Molecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events. Chitinase enzyme activity was detected using a standard enzymatic assay. The expression levels of chi26 gene in the different transgenic lines, compared to their respective controls, were determined using qRT-PCR. The transgene was silenced in some transgenic families across generations. Gene silencing in the present study seemed to be random and irreversible. The homozygous transgenic plants of T4, T5, T6, T8, and T9 generations were tested in the field for five growing seasons to evaluate their resistance against rusts and powdery mildew. The results indicated high chitinase activity at T0 and high transgene expression levels in few transgenic families. This resulted in high resistance against wheat rusts and powdery mildew under field conditions. It was indicated by proximate and chemical analyses that one of the transgenic families and the non-transgenic line were substantially equivalent. Conclusion Transgenic wheat with barley chi26 was found to be resistant even after five generations under artificial fungal infection conditions. One transgenic line was proved to be substantially equivalent as compared to the non-transgenic control.