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Backgrounds and Objectives: Classified as low pathogenic avian influenza (LPAI) viruses, the H9N2 subtype causes severe respiratory disease in poultry farms and occasional respiratory disease in humans. In this study, the neuraminidase (NA) gene of three Avian Influenza (AI) H9N2 strains isolated from poultry farms in Iran during 2010-11, as well as other reported Iranian H9N2 isolates, were genetically analyzed and their nucleotide changes were evaluated. Materials and Methods: The NA gene of three AIVs were sequenced and evaluated for genetic characteristics and phylogenetic relationship. Results: One new potential glycosylation site (PGS) at amino acid position 306 was observed in one of the studied isolates (A/Chicken/Iran/N102/2011). Antigenic sites of NA in Iranian H9N2 isolates have varied in a yearly manner. The Iranian isolates can be divided into 2 main subgroups; 11-T like subgroup viruses isolated mainly during 1998-2004 and second subgroup viruses isolated during 2004-2009. Interestingly, the three studied isolates fell into a third subgroup. The nucleotide sequences of the three studied isolates showed high identity to recent Pakistani H9N2 isolates (94.5-97%) compared to former Iranian AIV isolates (89-94%). Conclusion: High frequency of substitutions in the NA gene of studied isolates in recent years and effects of those substitutions on the pathogenicity of AI virus highlight the need to continue surveillance of genetic characteristics of AIV H9N2 in Iran.