Acinetobacter baumannii infection has become one of the most serious public health concerns due to its extensive resistances to all classes of antibiotics. Colistin is one of a few antibiotics that are still effective, but resistant strains have also gradually emerged. In Thailand, colistin is being used extensively therefore its resistance situation is critical. Colistin exerts its action by binding to the negative-charged phosphate moieties of lipid A, the endotoxic component of lipopolysaccharides. Colistin resistance in A. baumannii has been linked to the alteration in lipid A structure, that is the negative charges are negated thus eliminating the binding affinity to colistin. Fourteen colistin A. baumannii resistance strains with MIC > 2 μg/ml were isolated in Siriraj Hospital during 2011. All were typed by random amplified polymorphic DNA (RAPD). The lipid A from resistance strains were extracted and subjected to the analysis using MALDI-TOF mass spectrometry which revealed that the basic structure of A. buamannii lipid A is the heptaacylated diphosphoryl lipid A (m/z 1910). The resistance strains exhibited the extra peaks at either m/z 2034, 2071 or 2194, which correspond to the additions of phosphoethanolamine, hexosamine or both to lipid A molecule, respectively thus eliminating the negative charges of phosphate. The pmrCAB operon whose gene products are responsible for the addition of phosphoethanolamine was sequenced. Multiple mutations were found in pmrC and pmrB accordingly. These results suggested that the resistance mechanism of Thai isolates was the modification by which the negative charges were eliminated by the addition of small residues and may subsequently lead to the reduction of colistin target.