Background <p>Due to the widespread and irrational use of antibiotics, the emergence and prevalence of carbapenem-resistant <i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>) have become a major challenge in controlling bacterial infections in hospitals. The <i>bla</i>_{<i>KPC−2</i>} gene located on mobile genetic elements has further complicated the control of resistant bacteria transmission.</p> Results <p>In this study, <i>K. pneumoniae</i> strains were isolated from blood cultures of patients. Using the Kirby-Bauer disk diffusion method, we found carbapenem resistance heterogeneity. The resistant subpopulation KPTA-R1 and the sensitive subpopulation KPTA-S1 were purified. Whole-genome sequencing revealed that the <i>bla</i>_{<i>KPC−2</i>} gene in KPTA-R1 was located on an IncFII plasmid (pKPC-R), within a composite transposon (PCTs) formed by two direct repeats of IS<i>26</i> elements. The structure was identified as IS<i>26</i>-<i>RecA</i>-IS<i>Kpn27</i>-<i>bla</i>_{<i>KPC−2</i>}-IS<i>Kpn6</i>-IS<i>26</i>. However, in KPTA-S1, a similar plasmid, pAR-S, lacked this segment. Sequence comparison analysis indicates that the deletion of this <i>bla</i>_{<i>KPC−2</i>} encoding sequence in this IncFII plasmid is associated with transposition activity mediated by IS<i>26</i>. Multi-sequence comparison of the plasmids showed that the IS<i>26</i> transposon facilitated the sequence polymorphism of these plasmids.</p> Conclusion <p>This study reveals the key role of IS<i>26</i>-mediated transposition activity, through homologous recombination, in the emergence of carbapenem resistance heterogeneity in clinical <i>K. pneumoniae</i> strains carrying <i>bla</i>_{<i>KPC−2</i>}. IS<i>26</i> is able to promote the evolution of resistance in the IncFII plasmid, and through copy-in cointegration or targeted conservative cointegration may result in the acquisition or loss of antibiotic resistance, which may affect clinical care and pose a public health risk.</p>