Russian wheat aphid, Diuraphis noxia (Kurdjumov), was introduced into North America in Mexico during 1980 and by 1988 had spread north across the Western US into Southern Canada. South Africa has been suspected as the source of the introduction. Russian wheat aphid is the major insect pest of wheat and barley in the southern and central plains states. Resistant wheat with the Dn4 gene was the primary management tactic from 1996 to 2003. During 2003, a biotype able to injure Dn4 wheat (designated RWA2) appeared in Colorado. The source of the biotype is unclear, that is whether it represents a new introduction from another country or if it arose from the extant population in the US. We examined a 332 bp portion of the cytochrome oxidase subunit I gene (COI) in the mtDNA of Russian wheat aphid collected in the US from 1986 through 2006. Also included were specimens from Mexico and South Africa. Except for two nucleotides in two individual aphids, no COI sequence variation was found in US populations over a 20 yr period. US populations had COI sequences which were identical to those found in Mexico, Turkey, France, and one aphid from South Africa. A second aphid from South Africa differed by only two bp. The lack of COI nucleotide variation suggested that biotype RWA2 did not represent a second introduction of a genetically distinct population from another country. The data are consistent with a single introduction from South Africa and no other introductions of virulent biotypes into the US occurred since 1986.