This is a Real-Time quantitative PCR based chicken gender determination protocol to identify the sex of fertilized egg at the early chicken embryo states. It is based on the differences between sexual chromosomes: ZZ for males and ZW for females. Two pairs of primers have been developped to give a robust differential signal between males and females: Xho-I and DMRT.This protocol has been designed to sex chicken embryos in an invasive manner by openning the eggs.