A chromosome microdissection and microisolation technique in combination with filter hybridization was developed for chromosomal localization of cloned chicken genes. The DNA was obtained from microdissected chromosome regions of metaphase spreads. Dissected DNA was amplified by polymerase chain reaction (PCR). The chicken MHC gene located on the nucleolar chromosome and β-actin gene located on chromosome 2q were chosen as tests for the procedure and then detected by dot blot analysis using amplified chromosomal DNA probed with biotinylated DNA. The study establishes the technique of using chromosome microdissection and microisolation for localization of cloned genes as a complementary or alternative approach to both in situ DNA/chromosome hybridization and fluorescent in situ hybridization.