Shortly after its arrival, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), became established as the most important insect pest of soybean, Glycine max L. (Merr.), in the northern part of the North American soybean production region. Soybean resistance is an environmentally sustainable method to manage the pest and new soybean aphid resistant cultivars are beginning to be deployed into production. However, an earlier study identifying a soybean aphid biotype that could colonize plants with the Rag1 resistance gene has raised concerns about the durability of soybean aphid resistance genes. Choice and nonchoice tests conducted in this study characterized the colonization of a soybean aphid isolate, collected from the overwintering host Frangula alnus P. Mill in Springfield Fen, IN, on different aphid resistant soybean genotypes. This isolate readily colonized plants with the Rag2 resistance gene, distinguishing it from the two biotypes previously characterized and indicating that it represented a new biotype named biotype 3. The identification of soybean aphid biotypes that can overcome Rag1 and Rag2 resistance, even before soybean cultivars with the resistance genes have been deployed in production, suggests that there is high variability in virulence within soybean aphid populations present in North America. Such variability in virulence gives the pest a high potential to adapt to and reduce the effective life of resistance genes deployed in production. The search for new soybean aphid resistance genes must, therefore, continue, along with the development of alternative sustainable strategies to manage the pest.