Spread of an epidemic of avian influenza in 1998 in Iran was the cause of many deaths on farms. Research shows that made the emergence of this epidemic of avian influenza virus subtype H9N2. Anti-influenza drugs amantadine and rimantadine (Adamantane) were used for the treatment and prevention of infections caused by influenza A virus, but the use of these medications has been limited in recent years with increased resistance in the world. In a comparative study between the years 1998 to 2009 in Iran, to evaluate resistance to amantadine, the avian influenza A virus of subtype H9N2, we focused our research on the genetic structure of viruses, amantadine where most of their leaves left on the channel protein M2. Two groups of avian influenza A virus H9N2 subtype, were isolated in eggs with embryos, including five samples of the virus to two periods (1998-2006 and 2007-2009), then were the polymerase chain reaction tests using reverse transcription (RT-PCR) and electrophoresis on agarose gel and were sent for sequencing. The M2 sequence analysis displayed in different separate parts of this sequence as to consensus sequence that was amino acid changes together. And all of resistant isolates contained the point mutations (Ser to Asn) S31N at position 31 that could confer resistance to amantadine. The study took place, to determine the effectiveness of awareness of avian influenza viruses to amantadine, was reported, amantadine resistance in H9N2 subtype avian influenza viruses in the years 2007 and 2009 in Iran. Therefore, it is recommended to avoid the arbitrary use of amantadine to treat and prevent infections from avian influenza viruses of H9N2 subtype in the farms. Furthermore, it seems practical tests of the drug influence invivo, can ensure effective use of the drug.