Biotypes of aphids and many other insect pests are defined based on the phenotypic response of host plants to the insect pest without considering their intrinsic characteristics and genotypes. Plant breeders have spent considerable effort developing aphid-resistant, small-grain varieties to limit insecticide control of the greenbug, Schizaphis graminum. However, new S. graminum biotypes frequently emerge that break resistance. Mechanisms of virulence on the aphid side of the plant-insect interaction are not well understood. S. graminum biotype H is highly virulent on most small grain varieties. This characteristic makes biotype H ideal for comparative proteomics to investigate the basis of biotype virulence in aphids. In this study, we used comparative proteomics to identify protein expression differences associated with virulence. Aphid proteins involved in the tricarboxylic acid cycle, immune system, cell division, and antiapoptosis pathways were found to be up-regulated in biotype H relative to other biotypes. Proteins from the bacterial endosymbiont of aphids were also differentially expressed in biotype H. Guided by the proteome results, we tested whether biotype H had a fitness advantage compared with other S. graminum biotypes and found that biotype H had a higher reproductive fitness as compared with two other biotypes on a range of different wheat germplasms. Finally, we tested whether aphid genetics can be used to further dissect the genetic mechanisms of biotype virulence in aphids. The genetic data showed that sexual reproduction is a source of biotypic variation observed in S. graminum.