Alpha-tocopherol is one of four tocopherol isoforms and has the highest vitamin E activity in humans. Most cultivated soybean seeds contain γ-tocopherol as the predominant form, and the ratio of α-tocopherol content to total tocopherol content (α-tocopherol ratio) is <10%. Three soybean accessions from Eastern Europe have α-tocopherol ratios of >20%. This higher content is likely due to mutations in the promoter region of the γ-tocopherol methytransferase-3 (γ-TMT3) gene. We surveyed a wild soybean germplasm collection and detected 16 accessions with stable seed α-tocopherol ratios of >20% under different growth conditions. The α-tocopherol ratios were greatly reduced when the plants were grown under cool temperatures during seed maturation, but increased to varying degrees at higher temperatures. Sequence analysis of the γ-TMT3 promoter of 11 of the accessions identified four haplotypes, one of which corresponded to that of cultivars with higher contents. These wild accessions can thus serve as novel donors for breeding cultivars with high α-tocopherol ratios and for better understanding the genetic basis of α-tocopherol synthesis in soybean.