Near infrared (NIR) spectrophotometer offers rapid, noninvasive, nondestructive, and high-throughput phenotyping of seed samples for use in agriculture and industry. In this study, a reflectance-based NIR spectrophotometer was calibrated and used for the isolation of desirable higher-oleic-acid peanut recombinants from single-seed-derived segregating populations at F 7 and F 8 generations. A calibration model was developed through partial least-square regression using wet chemistry data from 158 peanut genotypes. Desirable prediction for oil, palmitic acid, oleic acid, and linoleic acid in intact seed was obtained based on this calibration. It detected significant high correlations (r) and coefficient of determination (R 2 ) between the actual gas chromatography values and NIR predicted values of fatty acid profile in another 123 peanut genotypes that were generated from crosses involving a high-oleate mutant and Spanish bunch varieties with early maturity. From this recombinant single-seedderived progenies, 15 higher-oleate recombinants were isolated and later genotyped through an in-house developed polymerase chain reaction-based allele specific marker. The present study has generated high-oleate peanut recombinants with early maturity in Spanish bunch background. The breeding materials generated here will be evaluated for yield attributing traits at different locations in future.Keywords Allele specific PCR marker Á Arachis hypogaea L. Á Fatty acids Á High oleic acid Á Mutant Á NIR spectrophotometer J Am Oil Chem Soc (2018) 95: 113-121.