The English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae), is an important pest insect of wheat, Triticum aestivum (L.), in China. Grain aphid biotypes are necessary to breed aphid-resistant wheat varieties; however, none have currently been identified. Here, we describe a method to identify grain aphid biotypes and survey the aphid biotype variation in the wheat growth area of China. Clones of S. avenae were collected from 11 locations in China and used to establish culture populations. These populations were then used to assess the resistance of 12 wheat varieties. Based on resistance responses, seven differential hosts were selected to identify the biotype of S. avenue: Amigo, ‘Fengchan No. 3’, Zhong 4 wumang, JP1, L1, 885479-2, and ‘Xiaobaidongmai’. S. avenae was ultimately classified into five biotypes: EGA I, EGA II, EGA III, EGA IV, and EGA V. These methods provide a mechanism to detect the variation and evolution of grain aphids in different wheat-growing locations and also allow for selection of appropriate aphid-resistant germplasm for wheat breeding of commercial wheat cultivars.