This paper reports for the first time the application of Raman confocal microscopic imaging to determine the impact of chemical and spatial differences in Raman spectra of ovine m. semimembranosus (SM) on indicators of tenderness and the potential to classify SMs based on shear force values. Chemical images of integrated intensities were generated and analysed against traditional indicators of tenderness. Results indicated that using the tyrosine doublet at 853 cm−1, it was possible to classify SMs into categories based on tenderness and soluble collagen content (P < 0.05). A promising classification was also achieved for the classification of SMs based on sarcomere lengths (P < 0.05) using the α-helix (930 cm−1) signal. Despite integrating the intensities and reducing wavenumbers values used in the analysis, the Raman spectra were still complex and the impact of spectral overlap from multiple compounds with similar chemical composition will need to be determined by future research.