Abstract: 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.