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Stearic acid is the most important saturated fatty acid compound in soybean oil. A typical soybean cultivar has 2–3% stearic acid content of total oil. Recently, a new high stearic acid mutant Hfa180 was developed from gamma-ray irradiation with “Ilmikong.” The objectives of this study were to identify the mutant gene associated with high stearic acid content in Hfa180 and to develop a Kompetitive Allele-Specific PCR (KASP) assay for selection of soybeans with elevated levels of stearic acid in seeds. Target sequencing of the whole GmSACPD-C gene encoding D9-stearoyl-ACP-desaturase revealed that Hfa180 contained a three-base-pair deletion (GAG) at genomic positions 190 to 192 of the 1st exon of GmSACPD-C. The three-base deletion caused a variant with a single amino acid (glutamine) deletion at position 64 in the predicted GmSACPD-C amino acid in Hfa180, as well as an average 60 …