Alpha (α-) tocopherol is the most active component of vitamin E and is a highly desirable and heritable trait in soybean seed. The objectives of this study were to identify and validate regions of the soybean genome that correspond to tocopherol accumulation in seed. A mapping population (07-225) of 190 RILs was derived from a cross between Keszthelyi A.S. x 2355RR which identified that α-tocopherol expression in soybean seed was strongly associated with the Satt381 – Satt196 region of chromosome 9 (LG K) in lines with Keszthelyi A.S. in their pedigree. Two other F4:5 validation populations derived from the crosses Keszthelyi A.S. x PRO 2995R (population 07-223; 94 RILs) and Keszthelyi A.S. x 2355RR (population 07-226; 45 RILs) were used to validate QTL in literature. The markers Satt079 (Ch 6; LG C2), Satt440 (Ch 20; LG I) and Satt381 (Ch 9; LG K) were significantly associated with α-tocopherol expression in these populations.