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Soybean (Glycine max) oil normally contains 2%–4% stearic acid. Increased saturated fatty acid levels elevate the melting point of soybean oil and improve oxidative stability. Using X-ray irradiation, we isolated six high stearic acid mutants and investigated mutations in the gene encoding Δ9-stearoyl-ACP-desaturase (SACPD). Our analyses of the KK2 mutant revealed an approximately 155-kbp deletion in the GmSACPD-B region. We also identified a frameshift mutation in the KK24 mutant, corresponding with a base-pair deletion in the GmSACPD-C coding region. The other four mutants M2, M18, MM106, and MS827 also had 2.2–21.8-kbp deletions in the GmSACPD-C region. Thus, we designed co-dominant marker sets for KK2 and KK24 mutant alleles using multiplex polymerase chain reactions and the CEL I mismatch detection method, respectively. In addition, we quantified the degradation of GmSACPD-C mRNA with a single base-pair deletion and related it to nonsense-mediated mRNA decay and simultaneous reductions in GmSACPD-A and -B mRNA levels. This molecular mechanism may be exploited to introduce silent mutations in duplicate genes of soybean plants and other organisms.