For pasta production, the yellow colour, mainly caused by carotenoids, is a worldwide requirement. Hexaploid tritodeums are the amphiploids derived from the cross between Hordeum chilense and Triticum turgidum. They show a higher carotenoid content than their wheat parents. This work aimed to develop a non-destructive method for carotenoid content determination to assist the tritordeum breeding program. We assessed the ability of near infrared reflectance spectroscopy (NIRS) to predict carotenoid content in whole grains of tritordeum. In total, 285 samples were scanned by NIRS. After non-destructive NIRS scanning, the seeds were analysed for carotenoid content and a calibration equation was developed. It is characterised by a coefficient of multiple determination (R²) of 0.85. This equation was initially evaluated by cross validation showing an r² of 0.81 and a standard error of cross validation (SECV) of 1.49. It was further evaluated using external validation with a different set of samples not included in the calibration. This analysis showed an r² of 0.81 and a standard error of performance (SEP) of 1.51. This equation allows discrimination between low and high carotenoid content lines in a non-destructive way. These results constitute a substantial advance for tritordeum breeding programs whose final aim is to develop high carotenoid content tritordeums useful for durum wheat breeding.