Avian influenza virus subtype H9N2 has been circulating and several outbreaks reported in poultry since 1998 in Pakistan. Drug abuse in poultry is the common practice in low to middle income countries including Pakistan. Neuraminidase Inhibitors are the drugs that target its protein neuraminidase. Identification of amino acid substitution in the neuraminidase of low pathogenic avian Influenza H9N2 viruses is important. In our study, the susceptibility and resistance patterns H9N2 were analyzed towards NAIs. Virus was serially passaged in-ovo under increasing concentrations of Oseltamivir and subjected towards haemagglutination assay, molecular confirmation and sequencing. At four points E119V, D151E, E276D, R371K, significant amino acid substitutions due to increase selective drug pressure were observed. These substitutions are responsible in reducing susceptibility of virus towards NAIs. The compensatory mutations in HA gene were also checked associated with NA gene. At position 234 of HA protein, the mutation was heading from Q towards L from passage 3 and persist till passage 5, that indicates that the virus is gradually modifying its host specificity range. As the virus having L at 234 position indicates its zoonotic potential as it has binding affinity more toward α 2,6 sialic acid receptors. So there is a need of continuous surveillance and monitoring while using NAI drugs against field viruses.