Technical Abstract: Soybean [Glycine max (L.) Merr.] oil typically contains 2-4% stearic acid. Seed oil with 20% stearic acid would be useful for solid fat applications, both for its cooking properties and health benefits. Breeding lines with high stearic acid have been developed, but many suffer from agronomic problems. This study: 1) identifies a new source of high stearic acid; 2) determines its relationship with another high stearic locus; and 3) and presents molecular markers for its use in breeding. TCJWB03-806-7-19, a ‘Holladay’ mutant with high stearic acid, was crossed to two FAM94-41-derived lines which contained a point mutation in a seed-specific isoform of a Delta 9–stearoyl-acyl carrier protein-desaturase (SACPD-C). Fatty acid analysis was performed over two growing seasons with F2-derived lines and transgressive segregation for stearic acid content was observed. Sequencing of SACPD isoforms in TCJWB03-806-7-19 revealed the deletion of an ‘A’ nucleotide in exon 3 of SACPD-B which results in a protein whose final 28 amino acids are predicted to differ from Williams82 SACPD-B. Sorting Tolerant From Intolerant (SIFT) analysis revealed that this frameshift mutation may affect SACPD-B protein function. Allele-specific genotyping for the SACPD-C point mutation and SACPD-B nucleotide deletion was performed in both populations. Additive effects and R2 for stearic acid were +3.3 and .55 for SACPD-C and +1.9 and .19 for SACPD-B. Average stearic acid in lines homozygous for both mutations was 14.6%.