Polyurethane production using soybean oil as a raw material provides opportunities to develop novel technologies for the automotive and other industries. Soybean lines with altered fatty acid profiles tailored for polyurethane production have been developed at the University of Guelph. The objectives of this study were to: evaluate soybean lines with altered fatty acid profiles for agronomic and seed compositional traits; identify simple sequence repeat (SSR) markers associated with fatty acid quantitative trait loci (QTL); and, determine the influence of different rates of phosphorus (P) and potassium (K) on the fatty acid profile of a high linoleic soybean line, RG25. In 2010 and 2011, two locations in southwestern Ontario were planted with eight modified fatty acid recombinant inbred (RIL) lines, one high linoleic, two high-oleic, two low-linolenic lines, and seven check cultivars. Field data were collected on emergence, plant height, lodging, days to flowering, days to maturity and yield. Fatty acid content was quantified using Gas Chromatography (GC). The high oleic line MFA-1 yielded highest in both locations in 2010, outperforming the commercial cultivars OAC Wallace and OAC Huron. In 2010, significant differences among genotypes for all fatty acid contents were detected. Only palmitic and linolenic acid content had a significant genotype by location interaction, suggesting that genotype response depended upon the environment for these two fatty acids. SSR markers were identified that were significantly (LOD≥3.0) associated with palmitic acid content at both field locations in 2010, including Satt556, Satt_355, and Satt389. Satt389 was also significantly associated with stearic acid content in Woodstock, ON, oleic acid content in St. Pauls, ON, and linoleic acid content at both locations. Different concentrations of P and K did not have a significant affect on the fatty acid profile of the high linoleic line, RG25. Research findings from this study will be beneficial to plant breeders aiming to develop modified fatty acid profile soybean lines for a renewable production of polyurethane for the automotive industry.