The H3N2 vaccine strain (A/Panama/2007/99) for the 2003-2004 influenza season did not antigenically match the circulating A/Fujian/411/02-like H3N2 viruses and had reduced effectiveness against influenza outbreaks. A/Wyoming/03/2003, an A/Fujian-like virus, was recommended as the vaccine strain for the 2004-2005 season. A/Wyoming differed from A/Panama by 16 amino acids in the HA1 molecule. Reverse genetics was used to determine the minimal amino acid changes that were responsible for the antigenic drift from A/Panama to A/Wyoming. After substitutions of 2 of the 16 amino acids in the HA (H155T, Q156H), the A/Panama HA variant was antigenically equivalent to A/Wyoming as determined by hemagglutination inhibition and microneutralization assays using ferret postinfection antisera. Conversely, A/Wyoming containing the His-155 and Gln-156 residues from A/Panama was antigenically equivalent to A/Panama. These results indicated that only these two HA residues specified the antigenic drift from A/Panama to A/Wyoming; other amino acid differences between these two H3N2 viruses had minimal impact on virus antigenicity but impacted virus replication efficiency in eggs.