With the recent publication of several European decrees prohibiting the culling of male chicks of laying breeds, the development of in-ovo sexing methods has become one of the priorities of the laying hen production industry. Regardless of the in-ovo sexing methods used, whether imaging approaches or quantification of specific compounds in embryo-derived samples, the prerequisite for accurate and sensitive in-ovo sexing methods is to establish a correlation between the egg picture and the sex of the embryo. It is therefore essential to know the sex of the embryo and have protocols that are easy to handle and implement, while allowing the analyses of hundreds of samples from a wide variety of tissues and chicken breeds. Among these protocols, real-time polymerase chain reaction using lysed samples containing DNA is likely to be of major interest. A few articles using specific primers, different types of chicken samples and breeds have been published, all with advantages and limitations. Here we describe a new protocol using a pair of primers designed to target a KCMF1-like gene located on the W sex chromosome, while the KCMF1 gene is located on the Z sex chromosome. This pair of primers was tested on four different chicken breeds at day 13 and 14 of development, using various tissues (liver, muscle, feather, heart, gonad, yolk sac, and chorioallantoic membrane) and fluids (blood and allantoic fluid). After 14 days of incubation, the comparison between the phenotype of male or female gonads and the real-time PCR results was 100% consistent, regardless of the breed. Thousands of samples in total have been analyzed by real-time PCR to validate the primers. The protocol is rapid and accurate and can be useful to sex 13 to 14-day old embryos to verify the sex predicted after evaluation of gonad dimorphism, or when the phenotypic sexing is inconclusive.