Abstract: To determine the key sites responsible for drug resistance in H3N2 neuraminidase gene, H3N2 wild type and three mutation type (E119D+I222L，E119D and I222L) viruses were generated by reverse genetics; The reverse genetics viruses were stable after five passages in MDCK cells; Oseltamivir resistance was detected by analyzing the kinetics of the two types of viruses in MDCK cells in the absence or in the presence of oseltamivir at the indicated times. We successfully generated H3N2 wild type and mutation type viruses; The wild type had a similar virus titer with the mutation type E119D; The virus titer of E119D+I222L and I222L type were lower than the wild type; The mutation type E119D+I222L had a high oseltamivir resistance while the wild type, E119D type and I222L type were greatly inhibited by oseltamivir. These results indicated that double mutation (E119D+I222L) type H3N2 could induce a high level of oseltamivir resistance. Thus, two amino acids in H3N2 neuraminidase gene (119, 202) are the two key sites responsible for oseltamivir resistance.