Technical Abstract: An outbreak of low pathogenic H5N2 avian influenza (AI) occurred in Mexico in 1993, and the virus mutated to the highly pathogenic form in 1994-95. The highly pathogenic virus was contained, but the low pathogenic virus has remained endemic in Mexican poultry in some states and has spread to two surrounding countries (El Salvador and Guatemala), even though control measures, including vaccination since 1995, have been used. Because this is the first case of long-term use of AI vaccines in poultry, the Mexican-lineage virus presents us with a unique opportunity to examine the evolution of AI virus in the presence of vaccination pressure. We analyzed the coding sequence of HA1 subunit of HA and NS gene of 52 Mexican-lineage viruses that were isolated between the years 1993 and 2002. Phylogenetic analysis indicated the presence of multiple sublineages of Mexican-lineage isolates at the time vaccine was introduced. Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccine sublineage. Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages. In vivo vaccine protection studies further confirmed the in vitro serologic results that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates. These findings indicate that antigenic drift, which has not been observed in AI virus, is occurring in the Mexican-lineage AI viruses and the persistence of the virus in the field is likely aided by its extreme antigenic difference to the vaccine strain.