The use of free, extractable lipids found in bacteria for identification of bacterial species and subspecies is described. Bacteria have been found to differ sufficiently in their extracted lipid compositions to effect identification using thin layer chromatographic techniques. Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei have been distinguished in this manner. Lipopeptides specific to Mycobacterium avium subspecies paratuberculosis, but not to the closely related bacterium Mycobacterium avium subspecies avium have also been used as a basis for bacterial subspecies identification using mass spectrometry and seroreactivity. Mass spectrometric analysis of total bacterial lipids of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei, and mass spectrometric analysis of total bacterial lipids for Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium, without further lipid separation, has shown that species and subspecies of bacteria may be identified using such analysis.