Multidrug-resistant infection has recently emerged as a worldwide clinical problem, and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance (HR) to colistin have been described. The purpose of the present study was to investigate the role of the PmrAB regulatory pathway in laboratory-selected mutants representative of global epidemic strains. From three unrelated clinical strains (sequence types 2, 3, and 20), eight colistin-resistant mutants were selected. Half of the mutants showed HR to colistin according to the reference method (population analysis profiling), whereas the other half exhibited stable resistance. M12I mutation within and M308R, S144KLAGS, and P170L mutations for were associated with HR to colistin, while T235I, A226T, and P233S mutations within were associated with stable resistance. The transcript levels of the operon were upregulated in all the mutants. Compensatory mutations were explored for some mutants. A single mutant (T235I mutant) displayed a compensatory mutation through IS mobilization within the gene that was associated with the loss of colistin resistance. The mutant resistance phenotype associated with T235I was partially restored in a -complementation assay turning to HR. The level of colistin resistance was correlated with the level of expression of in the -complemented strains. This report shows the role of different mutations in the PmrAB regulatory pathway and warns of the development of colistin HR that could be present but not easily detected through routine testing.