The soybean aphid, Aphis glycine Matsumura, is a new invasive insect herbivore, which is native to eastern Asia. Soybean aphid was first reported in the Midwestern US in July 2000. Resistance to soybean aphid was found in germplasm accessions and the dominant resistance gene Rag 1 in the cultivars Dowling and Jackson was mapped to soybean linkage group M. Extensive cultivation of soybean cultivars that have a single resistant gene over a large area of USA can result in rapid development of virulent soybean aphid biotypes capable of overcoming host plant resistance. But up to now, there have been no reports on whether major or minor biotype variation exists in soybean aphid in the USA. Choice and nonchoice tests were conducted to compare the virulence patterns of a soybean aphid biotype collected in Ohio with an isolate from Illinois on known aphid resistant and susceptible genotypes. There were significant differences in aphid reproduction on plants according to the aphid biotypes. The choice and nonchoice tests demonstrated that the Ohio soybean aphid biotype overcame the resistance gene Rag1 while the Illinois isolate did not. However, two soybean genotypes, PI200538 and PI 567597C, showed strong antibiosis and antixenosis to both soybean aphid biotypes. This newly characterized virulent soybean aphid isolate from Ohio poses a distinct threat to proposed soybean cultivars possessing Rag1. The two soybean genotypes that have resistance to the new soybean aphid biotype will be used to identify new aphid resistance genes and develop aphid resistant cultivars.