Abstract. The aim of the present study was to investigate the effect of in ovo feeding (IOF) of glutamine on hatchability, development of the gastrointestinal tract, growth performance and carcass characteristics of broiler chickens. Fertilized eggs were subjected to injections with glutamine (Gln) (10, 20, 30, 40 or 50 mg dissolved in 0.5 mL of dionized water) on day 7 of incubation. Hatchability, growth performance, carcass characteristics (carcass weight and relative weights of breast, thigh, heart, liver, gizzard, abdominal fat, intestine, pancreas and spleen) and jejunal morphometry (measurement of villus height and width and crypt depth) were determined during the experiment. The weight of newly hatched chickens was significantly greater in groups with Gln injection than in control and sham groups. But IOF caused lower hatchability than in the control group (non-injected eggs) (p < 0.05). Chickens from IOF of Gln showed better weight gain and feed conversion ratio (0–42 days of age), when compared to chickens hatched from control and sham groups. The IOF of Gln significantly increased villus height, villus width and crypt depth at hatch period and villus height at 42 days of age. In addition, carcass weights and relative weights of breast, thigh and gizzard were also markedly increased in chickens treated in ovo with Gln; whereas heart, liver, abdominal fat, intestine, pancreas and spleen were not significantly altered at the end of the experimental period. These data suggest that the IOF of Gln may improve jejunum development, leading to an increased nutrient assimilation and consequently to greater performance in broiler chickens.