In 2004, South Africa experienced its first recorded outbreak of a highly pathogenic notifiable avian influenza (HPNAI) viral strain of the H5N2 subtype in ostriches in the Eastern Cape province. The traditional ostrich-farming areas in the Western Cape province report almost yearly outbreaks of low pathogenicity avian influenza (LPAI) in ostriches, which is attributed to introduction by wild birds and certain climatic patterns. During the winter of 2004, LPAI H3N8, H4N8, H5N2 and H5N1 avian influenza viruses were isolated from wild aquatic birds. All eight genes of the H3N8, H4N8 and H5N1 viruses were analysed. The results show that the H5N1 virus does not belong to the HPAI Z/Z+N genotype currently circulating in Asia, but that the most recent common ancestors are Russian H5N2 and H5N3 viruses. The N1 gene lacks the stalk deletion associated with virulence. Internal genes probably originate from a pool containing Chinese, Middle Eastern and Italian viruses. The South African H3N8 and H4N8 viruses appear to have derived their genes from an ecosystem where Asian H5N1, H6N9 and H9N2, Russian H4, and Danish H3N8 viruses have been circulating since 1997. All three viruses share recent nucleoprotein common ancestors with the German and Dutch HPNAI H7N7 viruses from 2003. The diverse pool of genes from which local viruses are derived suggests that reassortment occurred at the Siberian breeding grounds where migratory paths cross, or within the South African ecosystem. This data highlights the importance of surveillance in aquatic migratory birds, particularly members of the Charadriidae, for their potential roles in the introduction of avian diseases to South African poultry and especially ostriches in the case of avian influenza.