Antiviral susceptibility screening of avian influenza (AI) H9N2 viruses is crucial considering their role at the animal-human interface and potential to cause human infections. The Matrix 2 (M2) inhibitors (amantadine and rimantadine) have been used for prophylaxis and treatment of influenza A virus infections, however, resistance to these drugs has been widely reported. Information about amantadine susceptibility of H9N2 viruses from India is scanty. Matrix genes of 48 H9N2 viruses isolated from India during 2009-2017 were sequenced and M2 trans-membrane region sequences were screened for mutations which are known to confer resistance to amantadine namely, L26F, V27A, A30 T/V, S31 N and G34E. All the viruses isolated during the year 2009 were sensitive to amantadine. However, resistance started to appear since the year 2010 and all the viruses isolated from the year 2015 onwards showed presence of molecular markers conferring resistance to amantadine. Majority of the resistant viruses exhibited S31 N mutation. Four isolates showed presence of V27A + S31 N dual mutations. Comparison of the M2 sequences from other Asian countries showed different patterns of amantadine resistance wherein phylogenetic analysis of the M genes of the strains from Pakistan formed a separate cluster. In conclusion, the present study reports prevalence and gradual increase of amantadine resistance among AI H9N2 viruses in India, emphasizing the importance of the antiviral surveillance.