A novel variant of the canine influenza virus H3N2 (cH3N2), designated as the M variant, was identified as containing a matrix (M) gene segment derived from the 2009 pandemic H1N1 virus (pdmH1N1), raising concerns regarding potential changes in antiviral drug sensitivity. In vitro and in vivo antiviral susceptibility assays demonstrated that while the parental cH3N2 strain was sensitive to amantadine, the M variant strain had acquired resistance to this drug. In addition, both strains remained susceptible to neuraminidase (NA) inhibitors such as oseltamivir and zanamivir. Comparative amino acid sequence analysis of the M2 protein identified a substitution, L22S, which is uniquely presented in amantadine resistant strains. Structural modeling of the M2 ion channel also suggested that the amantadine resistance observed in the M variant results from conformational alterations that impede drug binding. Collectively, these findings indicate that genetic reassortment with pdmH1N1 confers amantadine resistance in cH3N2 through the L22S substitution in the M2 protein. In addition, the preserved susceptibility to NA inhibitors suggests that these agents remain effective alternatives for controlling resistant strains, emphasizing the importance of continued molecular surveillance and diversified antiviral strategies.