Background <p>Antimicrobial resistance is a global concern. Tigecycline (TGC) and polymyxin B (PMB) constitute some of the last treatment options remaining for carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP). However, heteroresistance to these two drugs has rarely been reported. In this study, we investigated the prevalence of heteroresistance to TGC and PMB among clinically isolated CRKP strains, and assessed the synergistic activity of TGC plus PMB, TGC plus imipenem (IPM), and PMB plus IPM against heteroresistant CRKP.</p> Methods <p>A total of 90 nonduplicated CRKP clinical isolates were collected from Tianjin Medical University General Hospital, China,from January 2022 to October 2022. PCR was used to detect the resistance genes. Population analysis profile (PAP) was performed to determine the existence of heteroresistance. Checkerboard assay was carried out on ten randomly selected dual-heteroresistant CRKP strains. Additionally, MIC₅₀ (minimum inhibitory concentration) and MIC₉₀ were calculated, and the concentration-cumulative inhibitory percentage curves were plotted. Time-kill assays were conducted with selected CRKP strains.</p> Results <p>Among them, 48 (55.2%) of the 87 TGC-susceptible and TGC-immediate isolates exhibited heteroresistance phenotypes, and 61 (70.9%) of the 86 PMB-susceptible isolates showed heteroresistance phenotypes. Thirty-two isolates (35.6%) were heteroresistant to TGC and PMB simultaneously. PCR results showed that 96.7% (87/90) not only carried&#xa0;carbapenemase genes, but also co-harbored extended-spectrum β-lactamases (ESBLs) genes. Single time-kill curves demonstrated that PMB monotherapy could rapidly eradicate non-heteroresistant strains within 8&#xa0;h. Conversely, in heteroresistant strains, there was a sharp reduction in cells numbers initially but ensuing a prompt rebound. Intriguingly, further heteroresistance screening decoded the occurrence of heteroresistance phenomenon. Importantly, Checkerboard assay exhibited TGC combined with PMB exert relatively better effect to overcome dual heteroresistance compared with the other two regimes, with MIC₅₀ and MIC₉₀ of them were significantly declined than single drug and the concentration-cumulative inhibitory percentage curves shifted to the left. Moreover, they can early eliminate single heteroresistance in lower dose. Additionally, combined drug time-kill assays proved synergism effect. Overall, combining TGC and PMB may be a therapeutic approach to overcome counterpart heteroresistance in CRKP.</p> Conclusion <p>This study provides valuable insights and theoretical support for the clinical therapy of relevant heteroresistance.</p>