The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) /Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D144 mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens.