Key message <p>The combination of four variant fatty acid desaturate alleles achieves the desired high oleic and low linolenic acid fatty acid profile in soybean seed oil across U.S. production environments.</p> Abstract <p>Soybean is a global crop that represents over half of current world oilseed production. Soybean oil has been utilized in the food system based on the functional and health properties of the fatty acid profile; there is an urgent need to adjust the fatty acids in soybean oil to meet current market demands. The soybean high oleic acid seed oil trait (&gt; 75%) in conjunction with a low linolenic acid trait (&lt; 3%) has emerged as an avenue to improve the oil. The objective of this research was to identify a soybean genotypic group comprised of variant alleles of fatty acid desaturase genes that could meet the target stable seed oil fatty acid thresholds across the U.S. soybean production environments. New soybean genotypes were created with different gene combinations for seed oil fatty acid profile and maturity groups, seeds were produced in their target environments, and fatty acid profiles of the seeds were determined. The results demonstrated that a four-gene combination in soybean genotypes using either of two variant versions of one of the genes achieved both the target thresholds in environments across the U.S. soybean production regions. There were significant differences in individual fatty acid levels in the seed oil when comparing the two four-gene genotypic groups. The impact of this work is the new knowledge to design soybean varieties for U.S. production environments with seed oil fatty acid profiles targeted to meet current market thresholds; the fatty acid profiles obtained can serve as the basis for improved products.</p>