This experiment was conducted to investigate the effects of in ovo feeding and dietary feeding of betaine on performance, immunity, liver activity, blood cation-anion balance and bone parameters of chickens exposed to high temperatures. A total of 600 fertile eggs from a broiler breeder strain (Ross 308) were used for in ovo feeding of 0 and 1 g/L betainesolution at 17.5 d of incubation. After hatching, 192 male chicks were transferred to the experimental cages. The diets consisted of two types; one of them without betaine and the other one containing 1 g/kg of betaine. Then chicks were exposed to elevated temperatures 4 ˚C above optimum from 7-28 d of age for 4 h per d and after 28 d they were kept at optimum temperature. The chicks were divided into 4 treatments as a completely randomized 2 × 2 factorial design. There was no effect of in ovo feeding of betaine on performance, immunity parameters, liver activity, blood cation–anion balance and bone parameters. Chickens fed on the diet containing the betaine had the higher feed intake, daily weight gain and final body weight (p <0.05), but not significantly different feed conversion ratio. At 28 d, the weights of spleen and bursa of birds in the treatment groups fed betaine were higher (p <0.01). The betaine-fed group had higher antibody titer against infectious bursal disease (p <0.05), and lower heterophile/lymphocyte ratio, alanine aminotransferase and aspartate aminotransferase enzymes and C-reactive protein (p <0.01). In birds fed on the betaine in the diet, blood cation–anion balance was lower (p <0.01), whereas tibia length (p <0.05), and tibia ash percentage were higher at 28 (p <0.05) and 42 d (p <0.01). These results indicated that dietary feeding of betaine resulted in an improvement in performance, immunity parameters, liver activity, blood cation–anion balance and bone parameters and decreased the adverse effects of the high temperatures. Manuscript profile