Soybeans have diverse uses in foods and are known for their health-beneficial properties. Research has shown that consumption of soybeans or their components may help to prevent or alleviate chronic illnesses including heart disease, some cancers, and osteoporosis. Foods made from soybeans can develop flavors or odors that are unfavorable, in part because of lipid oxidation. Approaches to prevent lipid oxidation include modification of soybean traits such as fatty acid composition or lipoxygenase enzyme levels. Soybeans with modified α–linolenic acid (18:3n3) and soybeans with reduced lipoxygenase were analyzed to determine if there was an unintentional effect of modification on health-beneficial components. The effects of genotype and growing environment on nutraceutical components were analyzed for the modified 18:3n3 soybeans. Additionally, an assay for measurement of lipoxygenase–1content in soybean meal was developed for high-throughput analysis using fluorescein as a fluorescent probe. The results showed a significant effect of genotype of at least p ≤ 0.05 for fatty acid composition, isoflavone levels, lutein, and tocopherol levels in the modified 18:3n3 soybeans. Environment had significant effect on certain fatty acids, lutein, tocopherols, and oxygen radical absorbance capacity (ORAC). The effect of genotype × environment was significant for total phenolic content (TPC) and antioxidant activity against hydroxyl radical, as well as isoflavone composition, fatty acid composition, lutein, and tocopherol composition. Soybean lines with modified-lipoxygenase content contained similar or higher isoflavone levels compared to non–modified soybeans. The results of this research demonstrated that soybean modifications for improved oil stability did not adversely affect the health-enhancing components. Additionally, genotype or growing environment may be a factor in selecting the best soybean lines for nutraceutical development.