Influenza epidemics are a major health concern worldwide. Highly pathogenic avian influenza (HPAI) H5N1 viruses in Egypt have been subject to rapid genetic and antigenic changes since the first outbreak in February 2006 and have been endemic in poultry in Egypt since 2008. In this study, 33 H5N1 viruses isolated from avian hosts were antigenically analysed by using a panel of eight mAbs raised against the A/Viet Nam/1203/04 (H5N1; clade 1) and A/bar-headed goose/Qinghai-lake/1A/05 (H5N1; clade 2.2) influenza viruses. Rats were immunized with inactivated whole-virus vaccine produced by reverse genetics with the haemagglutinin and neuraminidase genes of eight antigenically different HPAI H5N1 virus isolates and six internal genes from A/Puerto Rico/8/1934 (PR8) to produce polyclonal antibodies. Cross-reactivity between the obtained polyclonal antibodies and the isolated viruses was assayed. Antigenic cartography of the isolated viruses showed that three antigenic clusters were defined based on haemagglutination inhibition (HI) analysis using mAbs and the majority of viruses isolated in 2010 and 2011 fell into two of these clusters. An antigenic map based on polyclonal rat antisera showed that all virus isolates fell within one extended cluster. Accordingly, continuous surveillance and antigenic characterization will help us determine which virus isolate(s) should be used in poultry vaccine preparation.