Previous studies have shown that the composition of glyphosate-tolerant soybeans (GTS) and selected processed fractions was substantially equivalent to that of conventional soybeans over a wide range of analytes. This study was designed to determine if the composition of GTS remains substantially equivalent to conventional soybeans over the course of several years and when introduced into multiple genetic backgrounds. Soybean seed samples of both GTS and conventional varieties were harvested during 2000, 2001, and 2002 and analyzed for the levels of proximates, lectin, trypsin inhibitor, and isoflavones. The measured analytes are representative of the basic nutritional and biologically active components in soybeans. Results show a similar range of natural variability for the GTS soybeans as well as conventional soybeans. It was concluded that the composition of commercial GTS over the three years of breeding into multiple varieties remains equivalent to that of conventional soybeans.