Since the introduction of Diuraphis noxia (Russian wheat aphid) in the United States in 1986, only one population existed. In 2003, a new population of the biotype appeared in Colorado, which overcame existing resistant cultivars. In subsequent years, at least six new populations were discovered in the Great Plains states. These newly emerged populations are referred to as biotypes, and are characterized by differential reactions to a set of wheat and barley genotypes containing different Russian wheat aphid resistance genes (Dn genes). The objective of this study was to analyze the genetic divergence among eight US biotypes and two South African biotypes using DNA markers. DNA markers used included simple sequence repeats (SSRs), randomly amplified polymorphic DNA (RAPD) markers, and amplified fragment length polymorphism (AFLP) markers. The mitochondrial cytochrome oxidase subunit I gene (mtCOI) was also completely sequenced and compared for nucleotide divergence among the US biotypes and South African biotypes. Over 12000 DNA fragments and mtCOI DNA sequences revealed limited polymorphism and nucleotide sequence divergence among the eight US biotypes. Phylogenetic trees established based on AFLP data and mtCOI sequence data show that the South African biotypes significantly differed from the US biotypes. The low level of genetic differentiation in both nuclear and mitochondrial genomes may be explained by founder effect, since the RWA species was just established in the US 20 years ago.