An H5-coinfection of a sample from wild ducks from which an H3N8 virus was isolated in 2004 was detected by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis of the partial H5 gene revealed that the strain was of low pathogenicity according to the amino acid sequence PQRTGLF at the Ho cleavage site. Phylogenetic analysis demonstrated that the partial H5 gene sequence fell within the American H5 lineage and shared 99.6% nucleotide sequence identity with the A/mallard duck/ALB/57/1976 (H5N2) strain isolated in 1976. Interregional transmission of influenza virus genes has been reported occasionally, although this is the first report from South Africa, where other influenza viruses have always had Eurasian origins.