This report examines lipophilic extracts containing mycolic acids isolated from tuberculosis (MTB) and non-tuberculosis (NTM) mycobacterial strains using chromatography, mass spectrometry (MS), nuclear magnetic resonance (NMR), and Raman spectroscopy. Gas chromatography-MS was used to identify major fatty acid mycolate components, while proton NMR confirmed the presence of characteristic cis- and trans-cyclopropane rings within different mycolic acid sub-types. Surface-enhanced Raman (SERS) spectra were obtained from the mycolic acids extracted from the bacterial cell envelopes of the MTB or NTM mycobacterial species. The Raman spectral profiles were used to develop a classification method based on chemometrics for identification of the mycobacterial species. Multivariate statistical analysis methods, including principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) of the SERS spectra enabled differentiation of NTM mycobacteria from one another with 100% accuracy. These methods are also sensitive enough to differentiate clinically-isolated MTB strains that differed only by the presence or absence of a single extracytoplasmic sigma factor with 83-100% sensitivity and 80-100% specificity. The current work is the first report on discrimination of mycobacteria strains based on the SERS spectra of the constituent mycolic acids in lipophilic extracts. These results suggest that SERS can be used as an accurate and sensitive method for species and strain discrimination in mycobacteria. © 2013 The Royal Society of Chemistry.