A simple, reliable, and safe protocol was developed for the collection of small amounts of blood from avian eggs of variable size and at early stages of development. Fifty eggs were used in the study; 40 were common chicken (Gallus domesticus) eggs, six were homing pigeon (Columba livia domestica) eggs, and four were burrowing owl (Athene cunicularia) eggs. Collection was attempted approximately halfway through incubation. The success rate for collection of blood or blood-tinged fluid from eggs was high, averaging 68% in the chicken eggs, 100% in the homing pigeon eggs, and 75% in the burrowing owl eggs. Collection did not affect subsequent hatchability. This blood could then be used to determine the sex of the embryo by utilizing a DNA probe or restriction fragment length polymorphism technique. Sex identification in ovo allowed the demographic management of small populations of birds within our institution.