Abstract: A microarray technique was developed by preparing highly conserved DNA fragments for five poultry respiratory tract diseases (Newcastle disease virus,avian influenza,infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma gallisepticum)using molecular cloning methods and the DNA fragments were spotted onto NC filter to form microarrays. RNA extracted from samples was reversetranscripted, then followed by PCR amplification and labling with biotin-11-dUTP.The labeled DNA and prepared DNA microarray were subjected to specific hybridization, the hybridization results were scanned and analyzed with a scanner. The results showed that the microarray technique could identify and distinguish the five poultry respiratory tract diseases. The method is rapid, sensitive and specific, and can screen or quarantine a large number ample samples within a very short time.