The complete nucleic acid and deduced amino acid sequences of gene segment 3 and the encoded VP3 from the North American, Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) are reported. Complementary DNA corresponding to segment 3 was 2768 nucleotides in length with an open reading frame of 2697 base pairs which encoded a VP3 polypeptide of 899 amino acid residues. Sequence comparison with genome segment 3 and VP3 from the Australian strain of EHDV-2 indicated genotypic and phenotypic homologies of 79% and 94%, respectively. Two North American field isolates of EHDV-2, as well as EHDV-1 (New Jersey isolate), had virtually identical homology to the Alberta isolate. Sequence analysis delineated North American EHDV strains as members of a genetically homologous and geographically distinct group of orbiviruses (topotype). The data support the hypothesis that geographic isolation between North American and Australian orbiviruses has permitted the viral topotypes to maintain their genetic distinctness.