We have developed a chicken (Gallus domesticus) Z-chromosome-specific DNA library in a phage vector by means of chromosome microisolation and microcloning. The chromosomal origin, specificity, and purity was evaluated by fluorescent in situ hybridization (FISH) on chicken metaphases. Heterologous chromosome painting using this Z-chromosome-specific probe on turkey (Meleagris gallopavo) metaphases identified its homologous Z-chromosome, under the same stringent conditions as that used in the chicken, indicating a high degree of Z-chromosome sequence homology among these two species. This chicken Z-chromosome library will facilitate the development of Z-chromosome-specific DNA markers that will be useful for genetic mapping in the domestic chicken and related avian species. The Z-chromosome-specific DNA probe will also be useful for studies pertaining to the sex chromosome evolution in avian species.