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Not AvailableThe objective of this study was to develop a simple and quick DNA extraction procedure for rapid diagnosis of sex of chicken and its embryos using multiplex polymerase chain reaction (PCR). Using alkaline method of DNA extraction from whole blood and feather bulb of adults, and tissue samples from embryos, the present study demonstrated that identification of sex by multiplex PCR protocol is simplest, safer, faster and inexpensive. Multiplex PCR was used to amplify W chromosome specific 481-bp fragment in female and 256bp fragment of 18S ribosomal gene in both male and female chicken. DNA samples were prepared by conventional phenol-chloroform-iso-amyl alcohol (PCI) method, modified PCI method, wizard genomic DNA purification kit and a simple alkaline method from blood samples and feather bulbs of adults and tissue samples of embryos. The protocol successfully identified sex of embryos and White Leghorn (Exotic), indigenous and Vanaraja (Hybrid of exotic and indigenous) varieties of chicken. Sequence comparison of W chromosome specific PCR products amplified from these three varieties showed no difference among them.SER