Abstract: Polymyxins, a class of cyclic peptide antibiotics, have become the last line of defense against gram-negative bacterial infections as multidrug-resistant bacteria continue to rise. Heteroresistance refers to the presence of subpopulations within the same strain with varying sensitivities to antibiotics, which cannot be detected by standard clinical tests and can result in treatment failure. In several common gram-negative bacteria, mutations in the PhoPQ and PmrAB two-component systems are key contributors to polymyxin heteroresistance. This review summarizes recent research on the mechanisms of polymyxin heteroresistance in gram-negative bacteria, providing insights for developing rapid detection methods and improving clinical treatment strategies.