Background & Objective: Quinolones are the antibiotics used to treat infections. Several reports have indicated the increased resistance to quinolone in Klebsiella pneumoniae strains all over the world. The aim of this study was to investigate amino acid substitutions in GyrA and ParC proteins among quinolone-resistant isolates of K. pneumoniae in Borujerd (west of Iran) hospitals. Materials & Methods: Totally, 100 isolates of K. pneumoniae were collected. After validating the isolates by conventional laboratory methods, an antimicrobial susceptibility test was carried out for some antibiotics from the quinolones family. Quinolone resistance and Minimum Inhibitory Concentration (MIC) of Ciprofloxacin were detected by disk diffusion and E-test methods, respectively. The amplification of gyrA and parC genes in quinolone-resistant strains was performed by PCR using specific primers. PCR products were sequenced in order to detect the mutations in gyrA and parC genes. Results: Generally, 38% of all the collected isolates were resistant to Nalidixic acid and Ciprofloxacin, 18% were resistant to Ofloxacin, and 15% were resistant to Norfloxacin. Concurrent resistance to Nalidixic acid, Ciprofloxacin, Ofloxacin, and Norfloxacin was determined in 15% of the cases. In 86% (n=20) of Ciprofloxacinresistant strains, MIC was measured 128 μg/mL. The mutation rate was 40% (n=9) in gyrA gene in quinolone-resistant strains and 35% (n=8) in parC gene. Conclusion: Briefly, our research findings revealed that a relatively high resistance to quinolones as well as fluoroquinolone was observed among K. pneumoniae isolates in Brojurd hospitals. It is likely that mutation occurrence in certain positions of gyrA and parC genes has a significant effect on developing high-level resistance to quinolone in K. pneumoniae.