DNA-based sex tests now exist for many avian species. However, none of these tests are widely applicable to ratites. We present DNA sequence data for a locus that is W chromosome-linked in the kiwi, ostrich, cassowary, rhea, and emu. At the amino acid level, this sequence has significant homology to X-linked genes in platyfish and Caenorhabditis elegans. Polymerase chain reaction (PCR) primers designed to this locus allow the assignment of sex in all species of living ratites.