An important goal for the development of high quality soybean oil is the reduction of the levels of polyunsaturated fatty acids and their substitution for the monounsaturated oleic acid. The main objective of this study is to improve the understanding of the underlying mechanisms and genes controlling oleic acid biosynthesis in soybeans. The desaturation of oleic to linoleic acid in seed triacylglycerols is encoded by the microsomal omega-6 desaturases, Fad2-1 and Fad2-2. The two isoforms of Fad2-1 gene are essential for the production of linoleic acid during seed development. Single nucleotide polymorphisms (SNPs) for the isoforms of Fad2-1 gene were detected in the mid-oleic plant introduction PI423893. A population of F₅-derived lines from the cross between the high-oleic, low-linolenic line N97-3363-3, and mid-oleic plant introduction PI423893 was grown in two locations in NC and phenotyped for oleate and other agronomic traits. Further insight on the allele-specific markers’ development and their association with several traits is provided. Future applications of this study in marker-assisted selection for the incorporation of the high-oleate trait in soybeans are discussed.