The fatty acid (FA) profile of beef is a key indicator of nutritional quality. This study assessed the ability of Near Infrared Spectroscopy (NIRS) to predict the FA profile in beef samples from southern Chile. A total of 81 FAs were analyzed, and 38% of the calibration models achieved RPD ≥ 2.5 (Ratio of Performance to Deviation). Strong predictive performance was observed for major FAs, particularly SFA and MUFA, with R²p > 0.90 (Coefficient of Determination) for palmitic (16:0). Although PUFA and some CLA isomers showed lower predictive accuracy—likely due to low concentrations and spectral overlap—minor FA such as 9c,11t-18:2 (CLA, rumenic acid) was accurately predicted. External validation confirmed that 77% of FAs showed no significant differences from gas chromatography, highlighting the robustness of NIRS for most compounds analyzed here. NIRS effectively captured FAs related to grass-based diets, such as trans-vaccenic acid and specific CLA isomers. NIRS works as a practical, rapid, and non-destructive tool for FA profiling, with potential uses in nutritional labeling and quality control; however, its application depends on the prior development of robust calibration models, which must be tailored to the specific matrix and analytical objectives.