This study evaluated the performance of classical front face (FFFS) and synchronous (SFS) fluorescence spectroscopy combined with Partial Least Square Discriminant Analysis (PLSDA), Support Vector Machine associated with PLS (PLS-SVM) and Principal Components Analysis (PCA-SVM) to discriminate three beef muscles (Longissimus thoracis, Recto abdominis and Semitendinosus). For the FFFS, 5 excitation wavelengths were investigated, while 6 offsets were studied for SFS. Globally, the results showed a good discrimination between muscles with Recall and Precision between 47.82 and 94.34% and Error ranging from 6.03 to 32.39%. For the FFFS, the PLS-SVM with the 382 nm excitation wavelength gave the best discrimination results (Recall, Precision and Error of 94.34%, 89.53% and 6.03% respectively). For SFS, when performing discrimination of the three muscles, the 120 nm offset gave the highest Recall and Precision (from 57.66% to 94.99%) and the lowest Error values (from 6.78 to 8.66%) whatever the algorithm (PLSDA, PLS-SVM and PCA-SVM).