BACKGROUND: Avian Influenza (AI) H9N2 subtype was first reported to have infected turkeys in the United States in 1966 and has been enzootic in Eurasia. In Iran, the H9N2 virus was first isolated from broiler chickens in 1998 in Ghazvin Province and it is the most prevalent subtype of influenza virus in poultry industry in Iran at the present time. The PB1 protein of influenza A viruses is an important host range and virus virulence determinant. OBJECTIVES: The purpose of this study was phylogenetic analysis of the PB1 gene of H9N2 AI virus isolated from broiler flocks in Iran during the period 1998-2011. METHODS: In this study, PB1 genes of 4 H9N2 isolates isolated from commercial chicken farms of Iran during the period 1998 to 2011 were partially amplified, sequenced and their amino acid sequences were assigned. The sequences were analyzed and phylogenetic study was done by comparing them to some deposited sequences of PB1 genes in GenBank. RESULTS: According to phylogenetic study on PB1 gene, two different groups can be distinguished among these Iranian H9N2 isolates. The current H9N2 circulating viruses in Iran are located in a new cluster of Middle East and India. The H9N2 isolates that are based on analysis of amino acid sequences of Iranian H9N2 isolates have some substitutions that are found in human and mouse adapted isolates. It seems that H9N2 isolates may show a trend to infect mammalian hosts. CONCLUSIONS: The available evidence indicates that PB1 genes of H9N2 influenza virus circulating in Iran have not been well conserved during the past years.