In spring 2003, several outbreaks of the Russian wheat aphid, Diuraphis noxia (Mordvilko), were reported in fields of supposedly resistant wheat cultivars (‘Stanton’, ‘Halt’, and ‘Prairie Red’) in eastern Colorado. We conducted two laboratory experiments to compare the biological performance of this new biotype 2 (B2) to that of two D. noxia collections of biotype 1 (B1) from western Kansas by using three wheat cultivars as host plants: ‘Trego’, a susceptible cultivar, and Stanton and Halt, two cultivars with different genetic sources of resistance. Survival of solitary nymphs from first instar to adult for the two clones of B1 on Trego was 96 and 90%, respectively, compared with 67 and 43% on Stanton, and 65 and 57% on Halt. In contrast, B2 had 60% survival on Trego, 43% survival on Halt, and 85% survival on Stanton. One clone of B1 required longer to mature on Halt compared with Trego or Stanton, but no other differences in developmental time among cultivars were significant. The standardized fecundity of solitary foundresses of the B1 clones was 19.6 and 20.1 nymphs on Trego, compared with 4.6 and 0.9 on Stanton, and 2.8 and 1.1 on Halt, respectively, over the same period. In contrast, fecundity of B2 was 21.1, 20.8, and 19.7 on Trego, Stanton, and Halt, respectively. When larger colonies developed on individual plants over longer periods, Trego supported the largest number of B1 aphids by experiment’s end, whereas Stanton and Halt yielded the largest numbers of B2. The order of overall plant damage was Trego > Stanton > Halt when infested with B1, with no significant differences for B2. Trego had more pronounced leaf rolling than other cultivars, independent of biotype. Collectively, the results suggest that D. noxia B2 from Colorado has evolved cross-virulence to both Dn4- and Dny-based resistance sources.