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Sesame is a crucial oilseed crop that contains vital fatty acids. The objective of this study was to build calibration equations using near-infrared reflectance spectroscopy for quality screening of sesame. A total of 136 sesame samples were scanned in the reflectance mode and their wet chemistry was determined by n-hexane extraction and gas chromatography mass spectroscopy. Models for oil and four fatty acids were developed with 110 samples and had an acceptable value of calibration coefficient of determination (R2 c), with suitable one minus the ratio of unexplained variance divided by variance (1-VR) value were found except palmitic acid. The prediction of an external validation with 26 datasets revealed a blameless correlation between reference values and NIRS values based on the coefficient of determination of validation (R2 v) and relative prediction deviation (RPD v). The models for oil, stearic, oleic, and linoleic acids had suitable values of coefficient of determination of validation and relative prediction deviation, which were more than 2.0 and 0.8, respectively. As a result of this research, it was discovered that the NIRS technology could be used to examine the oil and fatty acid contents of sesame seed qualities directly in breeding program, standard agency and commodity exchanges. • NIRS method reduces analysis time, non-destructive, and does not require solvents. • 110 samples for calibration and 26 samples for validation were used. • Second derivative and standard normal variate and detrend of spectral pretreatment were used. • The visible plus near-infrared region and modified partial least square was used to develop calibration equations. • NIR analysis of sesame could accurately predict oil content and three fatty acids. [ABSTRACT FROM AUTHOR]