Soybeans with a wide range of oil compositions were analyzed for their phospholipid (PL) fatty acid composition and stereospecific distribution. PLs were a minor component of soybean seed, comprising an average of 0.9% of the seed, which was an equivalent of 3.7% of the crude soybean oil. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were the major PL classes, with relative proportions of 55.3, 26.3, and 18.4%, respectively. These proportions were affected by the changes in oil fatty acid composition. The fatty acid composition of PLs changed with oil fatty acid modification. Stereospecific analysis showed that saturated fatty acids were primarily located at the sn-1 position of all PLs and unsaturated fatty acids were on the sn-2 position;PL compositional change may relate to the unpredictable and poor field seed performance. Soybean seeds with elevated and reduced percentages of palmitate and elevated percentages of stearate were compared with seeds of typical-composition in tests for germination, seedling growth rate, and leachate conductivity. In general, the seeds with altered compositions did well in these laboratory physiological tests, but their vigor tended to be more negatively correlated with the percentage of stearate than palmitate;The poor seed performance associated with the elevated saturate percentages may be attributed to the alteration in PL physical property caused by its compositional change. Neutral and polar lipids of modified soybeans were evaluated for their phase transition temperatures by differential scanning calorimetry. Neutral lipids with elevated stearate and both elevated stearate and palmitate had significantly higher melting temperatures than those with elevated palmitate, typical composition, and reduced saturates. PC from the elevated stearate lines and lines with both saturates elevated had significant higher phase transition temperatures than the other samples. Increased neutral lipid saturation may make it relatively unavailable as an energy source during seed germination. Increased PL phase transition temperature may cause cell membrane structural defects that affect membrane permeability and membrane-associated enzyme activities;PC and PE from the modified soybeans were partially fractionated into their molecular species by high-performance liquid chromatography. An evaporative light-scattering detector was used for quantification after its non-linear response was corrected. As expected, genetic modification of soybean oil caused changes in PL molecular fraction composition and this may have important implications in the physical properties and physiological functions of biomembranes.