This study examined the ability of near infrared reflectance spectroscopy (NIRS) to estimate the concentration of polyunsaturated fatty acids and their biohydrogenation products in the subcutaneous fat of beef cows fed flaxseed. Subcutaneous fat samples at the 12th rib of 62 cows were stored at -80°C, thawed, scanned over a NIR spectral range from 400 to 2498 nm at 31°C and 2°C, and subsequently analysed for fatty acid composition. Best NIRS calibrations were with samples at 31°C, showing high predictability for most of the n-3 (R(2): 0.81-0.86; RMSECV: 0.11-1.56 mg g(-1) fat) and linolenic acid biohydrogenation products such as conjugated linolenic acids, conjugated linoleic acids (CLA), non-CLA dienes and trans-monounsaturated fatty acids with R(2) (RMSECV, mgg(-1) fat) of 0.85-0.85 (0.16-0.37), 0.84-0.90 (0.21-2.58), 0.90 (5.49) and 0.84-0.90 (4.24-8.83), respectively. NIRS could discriminate 100% of subcutaneous fat samples from beef cows fed diets with and without flaxseed.