While soybean cultivars with resistance to the soybean aphid (Aphis glycines Matsumura) have been commercially released, the presence of virulent biotypes capable of overcoming plant resistance threatens the durability of host plant resistance as a stable management tactic. Novel sources of host plant resistance are needed to combat rapid biotype evolution. In this study, we screened 1,061 soybean plant introductions (PIs) for resistance to three known biotypes of A. glycines. Based on a series of growth chamber and field screenings, we identified 11 PIs that showed resistance to biotype 1 of A. glycines. Among these 11 PIs, 7 PIs were resistant to biotype 2 and 5 PIs were resistant to biotype 3. Further, two PIs (PI 606390A from Vietnam and PI 340034 from South Korea) showed resistance to all three biotypes of A. glycines. We also identified 11 PIs that were potentially tolerant to A. glycines, illustrated by no adverse impact on plant quality because of A. glycines infestation. As resistant PIs identified in this study belong to maturity group II–IV, they can be readily crossed to early maturing cultivars adapted to north-central states of the United States, where A. glycines is a major pest. The genetic characterization of resistance in these PIs and incorporation of novel resistant genes into elite soybean cultivars will broaden the defense against multiple biotypes of A. glycines.