Soybean with increased oleic acid is desirable to improve oxidative stability and functionality of soybean seed oil. Recently, soybean genotypes with high oleic acid (≥ 70%) were developed by conventional breeding and molecular assays. Determination of soybean genotypes with high oleic acid content has been done by gas chromatography and using DNA markers. Easier and effective determination of high oleic soybean genotypes using non-destructive near infrared reflectance (NIR) on whole seed would greatly enhance progress in development of high oleic soybean varieties. The objective of this study was to develop a calibration equation for NIR determination of high oleic acid from single soybean seeds. A total of 600 intact, single F₂ seeds were scanned by NIR.  Spectral data were collected between 400 and 2,500 nm at 2nm intervals. The relationship between NIR spectral patterns of each soybean seed and its oleic acid content was examined. The best predicted equations for oleic acid were selected on the basis of minimizing the standard error of cross validation and increasing the coefficient of determination. Validation demonstrated that the equations for determining seed oleic acid and high oleic acid content (over 50%) had high predictive ability (r²=0.91 and r²=0.99, respectively). To validate the newly developed equation, F₂ seeds from several different genetic backgrounds were tested. Again, high oleic acid from single soybean seeds was accurately predicted from various genetic backgrounds.  Therefore, applying the calibration equation to NIR will be useful to rapidly and efficiently select high oleic acid soybean genotypes in breeding programs