The generation of useful mutant alleles of specific genes would accelerate conventional breeding programs in various commercially important crops. Common soybean oil is easily oxidized because it is rich in polyunsaturated fatty acids (PUFAs). Microsomal omega-6 fatty acid desaturase (FAD2), which introduces a second unsaturated bond into oleic acid, is a primary target for elevating oleic acid levels and reducing PUFA levels. The paleopolyploid soybean genome contains five FAD2 gene homologues, at least three of which (GmFAD2-1a, 2-1b, and 2-2a) are functional. In spite of their importance, very little genetic variation has been identified in these genes except in GmFAD2-1a, because fatty acid content is easily affected by environmental conditions such as temperature. Here we isolated novel mutant alleles of GmFAD2-1b from ethyl methanesulfonate-treated soybean mutant populations through Targeting Induced Local Lesions In Genomes (TILLING), a reverse genetic method. Evaluation of enzyme activity in a yeast heterologous expression system suggested that two mutant lines, ‘B12’ and ‘E11’, contain near-null and null alleles, respectively, of GmFAD2-1b. Furthermore, by combining GmFAD2-1a and GmFAD2-1b mutant alleles, we successfully generated soybean lines with >80% oleic acid content. TILLING could provide a practical method for expanding the genetic diversity of polyploid crops.