Determining the sex of a bird on the basis of its morphological characteristics is difficult in >50% of avian species; in nestlings, this rate is even higher. Given that male birds have two identical sex chromosomes (ZZ), whereas females are heterogametic (ZW), the development of a DNA-based sexing method has been widely investigated in several species. Griffiths et al. (1998) reported a simple DNA test using intronic size variation; however, this method cannot differentiate birds that have small or nonexistent size variations. We report a simple and novel DNA test for avian sex determination using a novel primer (p0) specific for the unique sequence of the chromohelicase-DNA-binding (CHD)-W gene. Use of a multiplex polymerase chain reaction (PCR) and simple agarose-gel electrophoresis with p0 and with the primers P2 and P8 described by Griffiths et al. (1998) revealed that the P0–P2 combination could amplify the partial CHD-W gene alone. Consequently, we found that p0 could be used to identify only the CHD-W gene. We anticipate that this multiplex PCR will be useful for universal avian sexing, regardless of the intronic size variation.