Technical Abstract: The ordinary strain of Potato virus Y (PVY), PVYO, causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. There is, however, a growing body of evidence that PVYO strain group is not uniform both genetically and biologically. A novel sub-strain of PVYO was recently reported, PVYO-O5, which is spreading in the U.S. and is distinguished from other PVYO isolates serologically, i.e. reacting to the otherwise PVYN-specific monoclonal antibody 1F5. To characterize this new PVYO-O5 sub-group, and address possible reasons for its continued spread, we conducted a molecular study of PVYO and PVYO-O5 isolates from a North American collection of PVY through whole genome sequencing and phylogenetic analysis. Forty-four PVYO isolates were sequenced, including 31 from the previously defined PVYO-O5 group, and subjected to whole genome analysis. PVYO-O5 isolates formed a separate lineage within the PVYO genome cluster in the whole genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVYO sequences separated into at least two distinct lineages on the whole genome phylogenetic tree. A more detailed phylogenetic analysis of a sequence fragment, nt 2,406-5,821, that is present in all recombinant and non-recombinant PVYO genomes revealed that PVYN:O and PVYN-Wi recombinants acquired their PVYO segments from two separate PVYO lineages, while the PVYNTN recombinant acquired its PVYO segment from the same lineage as PVYN:O. These data suggest that PVYN:O and PVYN-Wi recombinants originated from two separate recombination events involving two different PVYO parental genomes, while the PVYNTN recombinants likely originated from the PVYN:O genome via additional recombination events.