One objective of soybean breeders is to develop cultivars with elevated oleic acid content. Testing soybean seeds for oleic acid content is possible using gas chromatography (GC), however it is time-consuming and requires destructing the seeds. Using single seeds, we developed a near-infrared reflectance spectroscopy (NIR) calibration equation relating oleic acid measured with GC to oleic acid predicted by NIR. The slope of the regression line of oleic acid measured with GC on oleic acid predicted by NIR and the intercept was not different from zero. A set of 300 soybean seeds was used to calculate the NIR equation and an independent set of 100 soybean seeds was used for validation. This NIRS equation showed significant correlation between reference values and NIRS estimated values based on the SEP, r2, and the RSP of reference data to SEP. This research shows that NIR prediction of oleic acid using intact soybean seeds is accurate and rapid and should be especially useful for early generation screening.