Sacha inchi (Plukenetia volubilis) seeds are rich in oil that contains a high content of polyunsaturated fatty acids. For genetic improvement and breeding of Sacha inchi, a rapid and nondestructive method for determination of oil content in seeds is critical. In this study, time-domain nuclear magnetic resonance (TD-NMR) was employed to determine oil content in Sacha inchi seeds at different developmental stages, in comparison with the standard Soxhlet extraction method. A highly significant correlation (R = 0.988) between oil content determined by TD-NMR analysis and that measured by Soxhlet extraction was found. Compared with Soxhlet extraction, the TD-NMR analysis provides a more accurate determination of oil content in Sacha inchi seeds at the late period of oil accumulation. The results demonstrated that the TD-NMR analysis is a non-destructive and efficient method for reliable determination of oil content in Sacha inchi seeds at different developmental stages.