Cultivation of aphid-resistant soybean varieties can reduce yield losses caused by soybean aphids. However, discovery of aphid biotypes that are virulent on resistant soybean greatly threatens sustained utilization of host plant resistance to control soybean aphids. The objective of this study was to identify and genetically characterize aphid resistant soybean accessions in a diverse collection of 308 plant introductions in maturity groups (MG) I and II. In large-scale screening experiments conducted in the greenhouse, we identified 12 soybean accessions (10 aphid-resistant and 2 moderately resistant), including nine previously not reported for resistance against soybean aphids. Three accessions (PI 578374, PI 612759C, and PI 603546A) and the Rag3 resistant check (PI 567543C) were susceptible when infested with a high initial aphid level but resistant when infested with a low initial aphid level, a phenomenon termed as density-dependent aphid resistance. Six accessions (PI 054854, PI 378663, PI 578374, PI 612759C, PI 540739, and PI 603546A) conferred antibiosis, five (PI 438031, PI 603337A, PI 612711B, PI 437950, and PI 096162) conferred both antibiosis and antixenosis, while one (PI 417513B) had neither when tested in no-choice and pairwise choice experiments. Molecular genotyping of the 12 accessions using single-nucleotide polymorphism (SNP) markers linked to known aphid resistance (Rag) genes revealed that PI 578374 and PI 540739 did not have any tested marker variants and could potentially carry unreported Rag genes. Genome-wide association analyses for MG I accessions identified genomic regions associated with aphid resistance on chromosomes 10 and 12 for each level of initial aphid colonization.