| Summary: | The intake of foods with unbalanced ω-6/ω-3 ratios causes various health problems. Commodity soybeans generally have a ω-6/ω-3 ratio of 6–7:1. The recommended ratio in terms of health benefits is <4:1. This study aimed to identify the appropriate combination of mutant alleles that can reduce the ω-6/ω-3 ratio using three segregating soybean populations. F<sub>2</sub> individuals from each population were genotyped for three different alleles of microsomal delta-12 fatty acid desaturase 2 enzyme (<i>FAD2-1A</i>) and an allele of homeodomain-like transcriptional regulator (<i>HD</i>) genes, and their five major fatty acids were assessed. F<sub>2</sub> seeds carrying both <i>fad2-1a</i> and <i>hd</i> had slightly different ω-6/ω-3 ratios according to the different <i>fad2-1a</i> alleles. The <i>fad2-1a<sub>DEL</sub></i>, <i>fad2-1a<sub>S117N</sub></i>, and <i>fad2-1a<sub>W293STOP</sub></i> alleles combined with a <i>hd</i> allele resulted in ω-6/ω-3 ratios with a range of 1.9–2.7:1, 2.7–3.9:1, and 2.6–3.6:1 in soybean seeds, respectively. This study revealed that the induction of mutations in <i>FAD2-1A<sub>DEL</sub></i> and <i>HD</i> was the most efficient strategy to improve the ω-6/ω-3 ratio and elevate the ω-3 fatty acid concentrations in soybean seeds. These results provide useful information in soybean breeding programs to release a new soybean cultivar with a lower ω-6/ω-3 ratio and elevated ω-3 fatty acids, which can be a beneficial ingredient for soybean-based foods.
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