The first confirmed outbreak of highly pathogenic avian influenza virus infections in North America was caused by A/turkey/Ontario/7732/1966(H5N9), however the phylogeny of this virus is largely unknown. We performed genomic sequence analysis of 11 avian influenza isolates from 1956 to 1979 for comparison with A/turkey/Ontario/7732/1966(H5N9). Phylogenic and genetic analysis included these viruses in combination with all known full genome sequences of avian viruses isolated before 1981. Here we show that a low pathogenic avian influenza virus, A/turkey/Ontario/6213/1966(H5N1), that had been isolated three months previous, was the closest known genetic relative with 6 genome segments of common lineage encoding the PB2, PB1, PA, NP, HA, and NS proteins. The lineages of these genome segments included reassortment with other North American turkey viruses that were all rooted in North American wild waterfowl with the HA gene originating from the H5N2 serotype. The phylogenies demonstrated adaptation from North American wild birds to turkeys with the possible involvement of domestic waterfowl. The turkey isolate, A/turkey/Wisconsin/1968(H5N9), was the second most closely related poultry isolate to A/turkey/Ontario/7732/1966(H5N9), possessing five common lineage genome segments (PB2, PB1, PA, HA and NA). The A/turkey/Ontario/6213/1966(H5N1) virus was more virulent than A/turkey/Wisconsin/68(H5N9) for chicken embryos and mice indicating a greater biological similarity to A/turkey/Ontario/7732/1966(H5N9). Thus we have identified A/turkey/Ontario/6213/1966(H5N1) as the closest known ancestral relative of HPAI A/turkey/Ontario/7732/1966(H5N9)which will serve as a useful reference virus for characterizing the early genetic and biological properties associated with the emergence of pathogenic avian strains.