The ratio of oleic acid to the combined value of linoleic and α-linolenic acids determines the oxidative stability, and the ratio of linoleic acid to α-linolenic acid is the key to the nutritional value of soybean oil. The present study was conducted to identify genomic regions associated with oleic, linoleic, and α-linolenic acids in recombinant inbred lines (RIL), developed from LSb1 × NRC7, across 5 cropping years. These RIL were genotyped using 105 polymorphic SSR markers across soybean genome and analyzed for fatty acid composition. SSR markers, namely, Satt245 (LGp M), Satt556 (LGpB2), Sat_042 (LGp C1), Staga002 (LGp D1b), Satt684 (LGp A1), and AI856415 (LGpD1b) showed significant (P < 0.05) association with oleic acid for all the 5 years, though this association was weak in the years when the growing temperature during active seed formation stage was high. Quantitative trait loci (QTL) linked to Satt684 (LGp A1), Satt556 (LGp B2), Sat_042 (LGp C1), and AI856415 (LGp D1b) showed pleiotropic influence on the levels of unsaturated fatty acids. Complementation of favorable QTL from LSb1 and NRC7 generated 60% oleic acid and less than 4% α-linolenic acid RIL, stable across 5 cropping years. New SSR markers, namely, Satt245, AI856415, and Staga002 identified to be associated with different unsaturated fatty acids may be useful in improving the efficiency of markerassisted breeding for enhancing the monounsaturated to polyunsaturated fatty acids ratio of soybean oil.