The development of biotypes of the cereal aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae), was initially found only on wheat, but barley can also be critical in the process.To address this issue, S. avenae clones were collected on barley and wheat, genotyped with six microsatellite markers, and tested with 58 wheat/ barley varieties. Based on the virulence response profiles on different resistant wheat/barley varieties and three susceptible controls, six biotypes of S. avenae were identified. We developed a new system to distinguish between S. avenae biotypes by using only five barley/wheat varieties (i.e., barley: Dulihuang, Zaoshu No.3, Xiyin No.2; wheat: Zhong 4 wumang, 186-TM12-34). The unique virulence profiles of different S. avenae biotypes were further verified by testing their life-history traits (i.e., 10-d fecundity and total developmental time of nymphs) on the abovementioned five barley/wheat varieties. Among all the identified biotypes, biotype 1 was predominant, occupying over 82% of the total in each province. Biotype 5 was found only in Xinjiang, whereas biotype 6 occurred only in Zhejiang. The principal coordinate analysis with microsatellite data suggested apparently low genetic differentiation between biotypes 1 and 2. In most cases, extents of genetic divergence between different S. avenae biotypes could reflect differences in virulence response profiles of these biotypes, implying a genetic component for evolutionary relationships among these biotypes. Our study provides insights into the development and evolution of aphid biotypes, and a firm basis for clarifying the underlying genetic and evolutionary mechanisms.