Sphingolipids (SL) are both structural and functional lipids with potential health benefits. To determine the relationship between palmitate and SL contents, methods for column separation and quantification using high-performance liquid chromatography - evaporative light scattering detector were developed for glucosylceramide (GlcCer) and ceramide (Cer) in 15 soybeans lines in which palmitate content ranged from 3.7 to 40.3%. Significant differences were observed among lines for GlcCer (83.4-397.6 nmol/g) and Cer (8.4-20.7 nmol/g) contents on a dry weight basis (dwb). But these contents were not significantly affected by the palmitate content of soybeans seeds. To evaluate the SL and phospholipid (PL) contents during seed development, soybean seeds of three cultivars were harvested at 5-day intervals from 28 to 68 days after flowering. Averaged across cultivars, the contents (dwb) decreased from 51.4 to 22.2 nmol/g for Cer, from 522.8 to 135.8 nmol/g for GlcCer, and from 9.1 to 3.5% for PL percentage relative to the total lipid.