This study was conducted to evaluate the effect of in ovo feeding of cationic amino acids on hatchability, hatch weights, and organ developments in pigeon squabs. Two experiments were conducted in this study. Eggs in Exp. 1 were subjected to modification of in ovo feeding in pigeons. Optimal time was determined by checking amniotic fluid volume, and suitable length was confirmed through ink injection. Results showed that the optimum time of in ovo feeding was on d 13 of embryonic development, and the suitable injected length was 20 mm to reach the amniotic cavity of the embryo. Eggs in Exp. 2 were transferred to access in ovo feeding of cationic amino acids. A total of 75 fertile pigeon eggs was randomly distributed into 5 treatments of 15 replicate eggs. Treatments in Exp. 2 consisted of non-injected controls (Control), a sterile buffered solution (0.75% saline), or a cationic amino acid mixture (> 98.5% purity crystalline L-arginine, > 98% purity crystalline L-lysine, and > 98.5% purity L-histidine) containing 0.1, 1, or 10% concentration (Conc.), which were relative to their total content in the eggs, respectively. The crystalline amino acids were dissolved in 200 μL buffered solution prior to in ovo feeding. After hatching, hatch weight (HW) and organ weight (OW) of the squabs were measured immediately. In ovo feeding of cationic amino acids increased the proportions of yolk-free hatch weight to hatch weight (YFHW/HW) (quadratic P = 0.01), and those of OW to YFHW including the heart (quadratic P = 0.01), kidney (quadratic P < 0.01), and liver (quadratic P = 0.02) compared to the control group, and the levels of those ratios were maximized in the 1% Conc group. Also, a proportion of small intestine weight to YFHW improved (linear P = 0.02, quadratic P = 0.05) after in ovo feeding. The organ weight of the head, leg, heart, lung, kidney, proventriculus, pancreas, liver, and small intestine correlated with YFHW positively (0.4 < correlation coefficient < 0.8, P < 0.05). In conclusion, cationic amino acids injection into amnion can improve the embryonic development, which may be mediated by the increment of relative organ weight.