A method of determining the gender of a bird in ovo comprises detecting the presence or absence of an elevated level of a sex-related hormone in the extra-embryonic fluid of the bird egg, and then determining the gender of the bird within the egg from the presence of an elevated level of a sex-related hormone therein. In particular embodiments, the sex-related hormone is an estrogen. In other representative embodiments, the invention provides methods in which the extra-embryonic fluid is allantoic fluid. In other illustrative embodiments, the method can be carried out on chicken eggs prior to or during transfer of the eggs from incubator to hatcher.