The current study was performed to investigate the antibiotic resistance pattern, the prevalence of colistin resistance and its molecular mechanisms in clinical isolates of Klebsiella pneumoniae obtained from hospitalized patients in teaching hospitals in Isfahan, Iran. This cross-sectional study was performed during 2019–2020 at several teaching hospitals in Isfahan, Iran. All the K. pneumoniae isolates were screened against 14 antimicrobial agents based on standard disk diffusion method. Moreover, the minimal inhibitory concentration (MIC) of colistin was determined by the E-test strips. Also, PCR assay was carried out to detect genes encoding resistance to colistin, including mcr-1, mcr-2, pmrA, pmrB, and crrB. In the present study, a total of 79 strains of multidrug-resistant K. pneumoniae were isolated. Of these, 35 colistin-resistant clinical K. pneumoniae isolates were obtained. Antibiotic susceptibility pattern showed a high rate of antibiotic resistance to ceftazidime (94.9%), while, the lowest resistance rate was observed against tigecycline (2.5%). All colistin-resistant isolates were classified as extensively drug-resistant (XDR). PCR results showed that mcr-1 and mcr-2 genes were not detected, while, 10.1% contained pmrA and pmrB genes. In addition, the frequency of crrB gene among our isolates was 3.8%. Due to the fact that the last line of treatment for infections associated with K. pneumoniae is colistin, increasing resistance to this antibiotic causes many concerns and problems in the treatment of patients. Detection of colistin-resistant strains and reporting of occurrence of genes associated with this resistance can greatly help in the treatment of diseases.