We investigated the efficiency of DNA extraction and the accuracy of polymerase chain reaction- (PCR-) based sexing of DNA that was obtained from adult chickens and early chicken embryos. DNA samples were prepared using water-, ammonium-, and proteinase-mediated extraction. PCR-based sexing was performed by amplifying a 276-bp fragment of the 717-bp W-chromosome-specific XhoI repetitive sequence. In blood samples from adult chickens, the time required for DNA extraction using the three aforementioned methods was less than that required by the conventional method of using phenol/chloroform/isoamyl alcohol (PCI). Accurate PCR-based sexing was possible using DNA samples that were prepared using the ammonium and proteinase methods. PCR-based sexing was successful for DNA samples from a 2-day-old embryo and the small intestine, gonad, and kidney of a 7-day-old embryo that were extracted using the ammonium and proteinase methods. However, DNA from the gonads of the 7-day-old embryo could not be sexed reliably using the ammonium method. In summary, the ammonium and proteinase methods are simple and practical means of extracting DNA for PCR-based sexing, but the reliability of sexing after use of the ammonium method should be checked when DNA samples have not been used previously for sexing.