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To determine sequence characteristics and phylogenetic relationships of the nonstructural (NS) gene, the NS genes of 9 avian influenza (AI) viruses were sequenced and compared with other Iranian AI H9N2 viruses available in GenBank. The similarity among the earliest Iranian H9N2 isolate (A/chicken/Iran/ZMT-101/1998) and recent (2007–2011) studied isolates was low (94%). Some of the studied viruses revealed substitutions in RNA-binding and effector domains as well as antigenic regions of the NS protein. Recently studied isolates have a high similarity (97.4%-98.7%) with some Pakistani isolates (like A/chicken/Sawabi/NARC-2434/2006) in the NS gene. In the phylogenetic analysis, recently studied isolates composed a cluster separate from other Iranian isolates and were placed in the neighborhood of some Pakistani isolates. Hence, new Iranian H9N2 isolates may originate from Pakistani isolates. The NS protein of the Iranian H9N2 isolates differed from that of the Iranian highly pathogenic H5N1 viruses. The potential risk of the emergence of a pandemic virus because of the reassortment of the internal genes between H9N2 viruses and highly pathogenic AI viruses on one hand and the genetic and amino acid changes of AI viruses affecting their pathogenicity and immunogenicity on the other hand confirm the importance of continued surveillance of AI viruses.