A simple method was used to adapt a standard light microscope for the collection of quail Z chromosomes from mitotic-metaphase spreads. The microisolated chromosomes were subjected to proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed PCR (DOP-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis, and smears of DNA revealed that ranged in size from 200-1 400 bp, without any evidence of preferential amplification. The second-round PCR products were cloned into pBluescript plasmids to construct a Z chromosome-specific DNA library. The size range of the cloned inserts was 200-1 400 bp. Using inserted fragments from the library as probes, chromosome painting was performed on quail chromosomes. The results showed that Z chromosomes of quail were completely covered by strong signals and there were little signals on other chromosomes. It was indicated that inserted DNA of the library was specific to the Z chromosome of quail. The library can be used as chromosome painting probe to detect conserved syntenic groups on the chromosomes of other related species and study mechanisms of sex-chromosomes evolution in birds.