Background and Aims: Avian Influenza (AI) H9N2 subtype was first reported to infect turkeys in the United States in 1966 and has been panzootic 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. Materials and Methods: In this study, we sequenced and analyzed Nucleoprotein (NP) gene of six AI H9N2 isolates from broiler farms of different parts of Iran from 1998 to 2011 to show probable changes since first advent. Results: Results indicate that nucleotide homology among these isolates with NP genes is between 91.8% to 98.8%. The divergences between isolates have significantly been increased since 2007.Iranian AI H9N2 Isolates based on NP gene divided in two distinct clusters according to their isolation year. Group 1 is located in Y-439 clade and Group 2 is located in G1 Clade. Iranian H9N2 isolates of avian influenza virus show more amino acid substitutions Compare to those found in human H9N2 isolates. Conclusion: The results shown here that further gene reassortment has occurred subsequent to the emergence of viruses in the Middle East highlights the potential for viruses to evolve rapidly.