Avian influenza (AI) is an infectious disease of birds caused by type A strains of the influenza virus. Among these AI viruses, only serotypes H5 and H7 are considered highly pathogenic in poultry. However, serotype H9N2 has also been found to produce severe respiratory tract infections in chickens. Migratory birds of the world are a natural reservoir of influenza viruses of all subtypes. This is the first report of isolation and molecular survey of AI virus from migratory and wild resident birds in Boushehr, Iran. These areas are at risk for the spread and transmission of influenza A viruses. A total of 443 fecal specimens (fresh droppings and cloacal swabs) were collected from migratory and wild resident birds in the Boushehr wetlands from October 2009 to June 2010. The AI virus was identified by reverse transcriptase polymerase chain reaction (RT-PCR) using a set of primers specific to the nucleoprotein of AI virus, and for positive samples, it was tested again by RT-PCR with specific primers for the H9, H7, and H5 subtypes. Low pathogenic AI viruses (H9 subtype) were detected by RT-PCR and virus isolation, but no highly pathogenic viruses were found during the period of study. The 1st and 2nd positive cases were in slender-billed gulls (Larus genei) from resident birds in the Helleh wetlands, and the 3rd positive case was in a mallard (Anas platyrhynchos) hunted on the Helleh wetland.