Soybean is one of important oil crops in the world, and improved linolenic contents of soybean oil will provide increased stability of soybean oil to the consumers and food manufacturers and limit the amount of trans-fat to be used in the processed foods. The linolenic content in soybean seeds is controlled by three fatty acid desaturase (FAD) 3 enzymes, including FAD3A, B and C, with FAD3A enzyme having the major effect on the final linolenic acid content in soybean seeds. The widely used soybean lines with reduced linolenic acid content for breeding in the U.S. have mutations in each of the three FAD genes including A5 (deletion of FAD3A), A26 and A23 (missense mutations in FAD3B and C, respectively). Although soybean line A5 has been released for 30 years, the deletion of FAD3A gene is unknown which prevented us from designing robust markers for effective marker-assisted selection. Using a PCR-based genomic strategy, we have identified a 6.5 kbp deletion of the FAD3A gene in A5 and developed a TaqMan detection assay by targeting the deletion junction in A5 which could be used to distinguish the homozygotes and heterozygotes of the gene. In addition, based on mutant SNPs in FAD3B and FAD3C identified in A26 and A23, respectively, we have also developed TaqMan assays for high throughput marker-assisted selection (MAS).  The TaqMan assays have proved to be a very effective platform to detect the mutant FAD3 alleles and thus will greatly facilitate the high-throughput MAS for development of soybean lines with reduced linolenic acid content.