BACKGROUND: Avian influenza virus H9N2 subtype and Ornithobacteriumrhinotracheale (ORT) causes histopathological lesions in birds. Yet, thereis not any study on tissue lesions caused by these pathogens co-infection inspecific pathogen free (SPF) chicks. OBJECTIVES: The aim of this studywas to evaluate the type, severity and extent of histopathological lesionsinduced by co-infection of H9N2 subtype avian influenza virus and Ornithobacteriumrhinotracheale in SPF chickens. METHODS: Sixty, one-day-old SPFchicks were divided randomly into three groups of twenty. At 21 days, thechicks in the first group were infected with H9N2 subtype AI virus and thesecond group was inoculated with H9N2 subtype AI virus and ORT simultaneously.The third group was inoculated with PBS as control. Then, three birds from eachgroup were randomly selected and euthanized and autopsied at 2  till 16 days post-inoculation (DPI). Thetissue samples were collected from trachea, lungs, liver, spleen, thymus, kidneys,cecal tonsil and bursa of fabricius. They were cut into 5 to 6 μm thicknesssections using paraffin embedding method and were stained by Hematoxylin andEosin (H&E). RESULTS: The severity of histopathological lesions inthe second group was higher than first group. Tissue changes were not observedin control group. CONCLUSIONS: The results of this study showed thatinfected chickens with H9N2 subtype AI virus and ORT simultaneously causeexacerbated histopathological lesions compared to H9N2 subtype of AI virus