In order to determine the causes of runny nose，sneezing，facial swelling and loss of appetite in a large number of broiler chickens in a farm in Yimen County，Yunnan Province，tissues such as nasal mucus，lungs and trachea of dead broiler chickens were collected to extract the nucleic acid of the pathogen after disposal，then to detect avian influenza virus（AIV），infectious laryngotracheitis virus（ILTV），Ornithobacterium rhinotracheale（ORT）and Haemophilus paragallinium（ICV），respectively by RT-PCR/PCR. The results showed that positive nucleic acid was observed only in ORT，and negative in all other pathogens. The sequence of 16S rDNA gene of the detected ORT positive samples was analyzed，and it was found that the 16S rDNA gene had more than 99.7% homology with the Iranian and Turkey/Hungary isolates，all of which were located in the same branch，which was similar to the information reported in China in 2014，indicating fewer pathogenic variations of ORT prevalent in Yunnan Province and a relatively short evolutionary history. Therefore，the study was of reference significance to guide the prevention and control of bacterial diseases in chicken flocks in the region.