Cyanogenic glucosides (CGs) are bioactive plant secondary metabolites that can release toxic hydrocyanic acid when hydrolyzed. The accumulation of CGs in flax seed is a safety issue as a feed component and may contravene international trade restrictions. Here, we report the identification of major effect quantitative trait loci (QTL) for linustatin and neolinustatin, the two most abundant CGs in the mature flax seed. Current methods of CG analysis for screening and quality control are prohibitively expensive. Here, we also report on the development of a low cost, high throughput method of analysis using near infrared spectroscopy (NIRS) to estimate individual CGs and total hydrocyanic acid equivalent based on a regression of reference data obtained by gas chromatography. The genetic and physical mapping of the QTL and the NIRS calibration hold direct applications in the development of a flax breeding strategy for developing germplasm and cultivars with reduced CG content.