This study aimed to evaluate theeffect of graded levels of substitution of corn with wheat and barley on performance, immunological responses and microbial population of different gastrointestinal segments in broiler chicks. A total of 420 one-day old Ross 308 broilers were used. Birds were randomly distributed between 7 treatments with 5 replicates. Dietary treatments consisted ofcontrol (based on corn and soybean meal), three different levels of wheat (15, 30 and 45 percent of the diet) and three different levels of barley (15, 30 and 45 percent of the diet) and were fed to chicken over 42 day. Between 23 and 27 days of age, birds were vaccinated against Newcastle disease, and one week after each immunization, blood samples were taken from birds to determine antibody titer. The blood samples after vaccination against Gumboro disease viruses and bronchitis, were analyzed. Between the ages of 27 and 34 days, sheep red blood cells (SRBC) were injected and one week after each injection, blood samples were taken from birds. At the age of 42 days, two birds from each cage were randomly weighted, killed and after dissection, internal organs and carcass were weighted. The contents of the crop, ileum and ceca of birds, were unloaded and traorted to the laboratory for microbial test. Results showed that dietary fiber source per starter, grower and finisher, and also the whole period, affected heavily on feed intake (FI)(P 0.01). Higher levels of the barley, significantly reduced feed intake, but even high levels of wheat had no effect on feed intake. As a result of decrease feed intake, daily gain of chickens was affected in each of the three periods and the entire production period (P 0.01). Feed intake and daily growth in chickens fed with high levels of the barley had the lowest growth. Adding wheat and barley increased conversion ratio. Especially, in initial period, barely linearly increased FCR (P 0.001). Carcass and the relative weight of abdominal fat decreased in response to adding wheat and barley to diets. Barley with high amounts of insoluble fiber significantly increased the relative weight of gizzard, ceca and the pancreas (P 0.01). Among lymphatic organs, the relative weight of bursa of fabricius was affected significantly (P 0.05). Thelowest relative weight of bursa of fabricius was observed in bird fed diet containing 45% wheat, while there was no significant difference between treatments. Blood triglyceride linearly decreased with increasing levels of wheat or barley. Also, by adding wheat and barley to diet, cholesterol significantly decreased compared to the control diet.Bacteria population was highly affected by adding wheat and barley in different areas of the gastrointestinal tract.So, the total bacteria in the cecum and Lactobacillus, Escherichia coli, salmonella and the total bacteria in the ileum were increased. In general, the results of this study showed that in region where maize cultivation is not possible, Wheat varieties with low viscosity can be a good substitute for corn. Also lower levels of the barley can used to developbetter digestive system, improve the efficiency of digestion and absorption of nutrients in the diet. Keywords: Broilers, Fiber, Cereal, Microbial population, Blood biochemical parameters Development of digestive system