Technical Abstract: Nineteen isolates of the cereal aphid pest greenbug were collected from wheat, barley or noncultivated grass hosts in five locations from the states of Colorado and Wyoming in the U.S., and parthenogenetic colonies were established. Biotypic profiles of the 19 isolates were determined based on their abilities to damage a set of host plant differentials and 13 new biotypes were identified. Genetic diversity among the 19 isolates and five previously designated greenbug biotypes (E, G, H, I, and K) was examined with 31 cross-species transferable microsatellite (simple sequence repeat, SSR) markers, and a phylogenetic tree was constructed. Neighbor joining analysis of the microsatellite marker data revealed host-adapted genetic divergence as well as regional differentiation of greenbug populations. Host associated biotypic variation seems to be more obvious in ‘agricultural biotypes’ whereas isolates collected from non-cultivated grasses tend to show more geographic divergence. The implications of these findings on the development of biotypes and breeding for greenbug resistance were discussed.