The effects of in ovo injection of 25-hydroxycholecalciferol [25(OH)D3] at various dosages on weight, breaking strength, and ash and mineral concentrations of left tibial bones in Ross × Ross 708 broilers on d 0 and 21 posthatch were investigated. Six treatment groups were equally represented on each of 10 tray levels (blocks) of a single-stage incubator. The 6 treatment groups, containing 21 eggs each, were randomly arranged on each tray level with respect to those on other tray levels. A multi-egg injector was used to inject eggs on d 18 of incubation according to their preassigned treatments. The experimental treatments included noninjected and diluent-injected (100 µL of commercial diluent) controls, and those that received 0.15, 0.30, 0.60, or 1.20 µg of 25(OH)D3 in 100 µL of diluent. On d 0 and 21 posthatch, 3 chicks from each treatment replicate group were randomly selected for necropsy and the subsequent extraction of their left tibial bones. Tibia samples were weighed and then subjected to breaking strength and ash concentration analysis. One of the 3 residual bone ash samples in each treatment replicate group was randomly selected, digested, and analyzed for Ca, P, Mg, K, Zn, and Mn concentrations. The relative weight, breaking strength, and percentage ash and mineral concentrations of broiler tibias on d 0 and 21 posthatch were not affected by treatment. However, trend analysis of percentage tibial ash on d 21 revealed a dosage effect (P = 0.068). The results of this study suggest that the in ovo injection of 0.15 to 1.20 µg of 25(OH)D3 on d 18 of incubation did not influence subsequent bone development or mineralization in Ross × Ross 708 broilers on d 0 and 21 posthatch. Nevertheless, further trend analysis suggested that increased dosage levels of 25(OH)D3 above 1.20 µg may have the potential to lead to increases in bone mineralization.