Soybean aphid (Aphis glycines Matsumura (Hemiptera: Aphididae)) has been a major pest of soybean in North America since its detection in this continent in 2000 and subsequent spread. Although several aphid resistance genes have been identified, at least four soybean aphid biotypes have been discovered, with three of them being virulent on soybean cultivars with certain soybean aphid resistance genes. These biotypes are known to vary across years and locations, but information on their variation within single fields is limited. An investigation was conducted to study the variation of soybean aphid biotypes within single townships and fields in Minnesota. Screening of 28 soybean aphid isolates collected from seven soybean fields (six soybean fields in Cairo and Wellington Townships of Renville County, MN and one field in Wilmar Township of Kandiyohi County, MN) revealed the existence of multiple known biotypes of soybean aphid within single fields of soybean. We found up to three biotypes of soybean aphid in a single field. Two biotypes were found in five fields while only one field had only a single biotype. Three isolates presented reactions on a panel of resistant and susceptible indicator lines that were different from known biotypes. These results highlight the importance of characterizing soybean aphid biotypes in small geographical areas and utilizing generated knowledge to develop soybean cultivars pyramided with multiple resistance genes. The outcome will be decreased use of insecticides, thereby improving economic and environmental sustainability of soybean production.