Total of 400 hatching eggs with an average egg-weight of 60.5 ± 1.5g from Hy-Line Brown Strain acquired from layer breeder flock (46 weeks of age) were used in this study to investigate the impact of in-ovo injection of omega-3 fatty acids on hatchability and some physiological and histological parameters of the developing chick embryos and newly hatched chicks. At the 10^th day of embryogenesis, fertile eggs were allocated into four equal groups. In the 1^st group, embryonated eggs did not receive any treatment and served as the control group. Eggs of the 2^nd, 3^rd and 4^th groups were injected into the yolk sac with 200μL sterile saline, 100 and 200µL of omega-3 fatty acids, respectively. Results revealed that in-ovo injection with low dose of omega-3 (100µl) resulted in a significant increase in the hatchability percent, embryonic and post-hatch body weight, serum total protein, globulin, thyroid hormones concentrations and total antioxidant capacity in the hepatic and brain tissues as compared with high dose of omega-3 (200µl), or control groups. While, significant increases in serum total lipids, cholesterol, triglycerides concentrations and alkaline phosphatase, alanine transaminase, and aspartate transaminase enzymes activities as well as harmful histological changes in hepatic tissues were observed in high dose of omega-3 (200µl). In conclusion, in-ovo injection with 100µl of omega-3 caused enhancement in hatchability, body weight, total antioxidant capacity of hepatic and brain tissues and some of the physiological aspects during embryonic development and at hatch.