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This study examined the impacts of four wheat cultivars and enzyme supplementation on growth performance, nutrient digestibility, and ileal microbiota composition in broiler chickens. Six hundred forty-eight male broilers (1-day-old, Ross 308) were studied in a completely randomized design factorial 4 × 2 along with control (9 treatments) with 6 replications (12 birds per pen). The Diets consisted of the four varieties of wheat (Sardari, Azar2, Sirvan, and Pishgam) with and without enzyme supplementation, alongside a corn-based control diet. All diets were iso-caloric and iso-nitrogenous. Daily weight gain (DWG) and feed conversion ratio (FCR) were not significantly affected by the dietary treatments. Broilers fed the corn-based diet displayed higher feed intake (FI) than those fed diets containing different wheat cultivars. Enzyme supplementation in wheat-based diets did not impact broiler growth performance. There was an interaction between enzyme and wheat type for protein, fat, calcium, and phosphorus digestibility. Ileal microbiota analysis revealed no significant changes in Lactobacillus and Escherichia coli populations across treatments. Conversely, Enterococcus and Bifidobacteria populations exhibited significant differences, with the Sirvan cultivar diet promoting the highest bacterial counts. It was concluded that different wheat cultivars could affect growth performance, nutrient digestibility, and ileum microbiota, and the beneficial effect of supplemental enzymes was only evident in certain variables and depended on the specific wheat variety.