A polymerase chain reaction (PCR) technique, targeting a chromo-helicase DNA-binding protein (CHD) gene, was used to determine gender in birds. DNA from 87 birds, representing 31 species from 9 of the 20 orders of birds, was evaluated. The DNA was extracted from as little as 5 μ l of whole blood, and CHD-specific primers (P2 and P8) were used to amplify the CHD gene. Polymerase chain reaction products were then subjected to restriction fragment length polymorphism analysis. Gender was determined by distinguishing between the female CHD-W gene and the male CHD-Z gene by agarose gel electrophoresis of the digested PCR products. The PCR assay using the HaeIII restriction enzyme helped identify gender in all species evaluated except those from the Order Anseriformes. Isolation of the CHD-Z and CHD-W fragments was performed, and the genes were cloned and sequenced in a number of waterfowl species. On the basis of sequence results for the CHD-Z and CHD-W genes for both the mallard duck (Anas platyrhynchos) and the Canada goose (Branta canadensis), the cleavage site for the enzyme EcoRI was identified, and the lack of a cleavage site for HaeIII was confirmed. EcoRI was then used to sex 5 duck species and the Canada goose.