The effect of in ovo administration of different levels of betaine and choline on egg hatchability, immune response, growth and carcass traits of broiler chickens was studied. Four thousand hatching eggs from Ross 308 broiler breeder layers, weighed individually, were incubated for 21 days in a commercial hatchery. At 12th day of incubation, 3456 fertilized eggs were randomly divided into 8 experimental groups of 3 replicates each (144 eggs per replicate): negative control (NC) – not injected; positive control (PC) – injected with 0.5 mL deionized water; Bet 0.25 – injected with 0.5 mL deionized water+0.25 mg soluble betaine; Bet 0.375 – injected with 0.5 mL deionized water+0.375 mg soluble betaine; Bet 0.50 – injected with 0.5 mL deionized water+0.50 mg soluble betaine; Chol 0.25 – injected with 0.5 mL deionized water+0.25 mg soluble choline; Chol 0.375 – injected with 0.5 mL deionized water+0.375 mg soluble choline; Chol 0.50 – injected with 0.5 mL deionized water+0.50 mg soluble choline. Among the hatched chickens, 360 males were randomly chosen (45 for each group) and were grown up to 42nd day of age. The embryo mortality, pecked eggs, infected eggs and hatchability percentages were similar among the experimental groups. The betaine and choline treatments improved hatching weight and final weight of chickens, while reduced feed conversion ratio and abdominal fat percentage. No effect on carcass yield, and breast muscle, leg and wings percentages, as well as on immunoglobulin M (IgM), G (IgG), and total antibody (IgT) titers was observed. The treatments had little effect on internal organs.