Background: M2 channel protein of influenza A virus is one of the specific targets for the anti-influenza drugs amantadine and rimantadine. These drugs have lost their efficacy because of the mutations in their drug interaction sites. Large-scale analysis of these influenza surface proteins may give better elucidation for understanding the evolution of the proteins toward the drug resistant mechanism.Objective: The current investigation aimed to understand the evolutionary lineage and to enlighten the mechanism of drug resistance in newly emerging strains. Method: Combined sequence, secondary structural, evolutionary conservation, and phylogenetic analyses were carried out with 2010 influenza A M2 channel protein sequences. Results: The structural information provides enough details for understanding the drug resistance in the target proteins. Herein, secondary structural analysis of M2 sequences predicted the variation only in the drug binding region. The rate of mutation in S31N is high in swine/H3N2 than in human/H1N1, human/H3N2, swine/H1N1, and avian/H5N influenza A viruses. This confirms that antigenic drift does not affect the functional mechanism of the protein. Also, it reports that the avian influenza virus is the source for the M2 gene segment and has transferred from the avian to human and swine. Our findings show that the M2 gene segment has interchanged between swine and human. Conclusion: This study proves that rapid mutation and frequent reassortment play a major role in drug resistant strains. Phylogenetic and secondary structural analysis confirms the existence of a genetic lineage between avian, swine, and human influenza A viruses.