Australian grass and grain-fed beef products attract premium prices at sale and several beef processors market beef underwritten by production system claims. This preliminary investigation assessed the feasibility of using Raman spectroscopy to detect differences in the chemical composition of subcutaneous fat from cattle raised in extensive and intensive production systems. Raman spectra, fatty acid composition, β-carotene composition and objective colour measurements were measured on 150 grass and 150 grain-fed cattle. Spectral differences at peaks including 1069 cm−1, 1127 cm−1, 1301 cm−1 and 1445 cm−1 suggest that Raman spectra is able to detect differences in saturated fatty acids, which were significantly higher in carcases from grain-fed cattle. Differences in spectra at 1658 cm−1 were observed, however further research is required to investigate the cause of this spectral feature. Overall, this study indicated that Raman spectroscopy is a potential tool for the authentication of beef carcases from grass and grain-fed production systems.