The aim of this research was to estimate the fatty acid (FA) content of intramuscular fat from beef by Fourier transform mid-infrared (FT-MIR) spectroscopy. Four diets were supplemented in 10% linseed (LS) and/or 2% conjugated linoleic acid (CLA): CON (without L or CLA), LS, CLA, and LS+CLA. For each diet, 12 young Holstein bulls were allocated. The spectral response of the beef samples was analyzed applying FT-MIR spectroscopy (from 400 to 4000 cm−1) and predictive models were developed using partial least square regression with cross-validation. The obtained coefficients (R2) for some FA, such as α-linolenic acid with a R2 = 0.96 or n-3 polyunsaturated fatty acids (n-3 PUFA) with R2 = 0.93, demonstrate that FT-MIR spectroscopy is a valid technique to estimate the content of FA. In addition, samples were correctly classified according to the animal diet using discriminant analysis in the region 3000–1000 cm−1. The obtained results suggest that the FT-MIR spectroscopy could be a viable technique for routine use in quality control because it provides fast and sustainable analysis of FA content. Furthermore, this technique allows the rapid estimation of the FA composition, specifically n-3 PUFA and CLA, of nutritional interest in meat. It also allows the classification of meat samples by the animal diet.
