Nineteen isolates of the cereal aphid pest greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), were collected from wheat, Triticum aestivum L.; barley, Hordeum vulgare L.; or noncultivated grass hosts in five locations from Colorado and Wyoming. 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) markers. Neighbor-joining clustering analysis of 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 noncultivated grasses tend to show more geographic divergence. It seems that the biotype sharing the most similar biotypic profiles and the same geographic region with current prevailing one may have the greatest potential to become the new prevailing biotype. Close monitoring of greenbug population dynamics especially biotypic variation on both crop plants and noncultivated grasses in small grain production areas may be a useful strategy for detecting potentially new prevailing virulent biotypes of the greenbug.