Molecular characterization of neuraminidase (NA) gene of 25 influenza A(H3N2) virus isolates (2009-2013) archived at the Manipal Centre for Virus Research was carried out. The annual rate of amino acid substitutions in the N2 gene of influenza A(H3N2) virus isolates was 0.2-0.6%. Out of the 25 NA sequences analyzed, catalytic site mutations were observed in three isolates. Two of the mutations (D151G and E276G) were detected in functional catalytic residues, and an E227V mutation was detected in the framework residues. To the best of our knowledge, NA inhibitor resistance associated with the mutations E276G and E227V has not been reported. However, the mutation D151G, which is commonly associated with culturing of influenza A(H3N2) virus in Madin-Darby canine kidney (MDCK) cells, has been reported to result in a reduction in virus susceptibility to NA inhibitor drugs. Our study also detected mutations in antigenic residues. Some of the mutations (except D197G, K249E, A250T, S334C, and H347R/N) remained conserved in isolates of succeeding seasons. Antigenic residue mutations (D197G and S334C) have not been reported globally to date. The effect of these catalytic and antigenic mutant residues on drug and antibody binding was analyzed using three-dimensional structural analysis and biochemical assays. Antigenic variability of influenza A(H3N2) viruses is a major concern, and vaccine failures are mainly due to genetic variations in the HA gene. Our study documents that genetic changes in N2 occur at a slower rate, and this information is useful for the consideration and standardization of NA in influenza vaccines.