The frequency of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli and Klebsiella pneumoniae clinical isolates is a serious issue in patients with leukemia. One of the critical factors implicated in this resistance manner may be genes encoding enzymes producing the protecting effect against quinolone-based antibiotics. The goal of this study was to determine the prevalence of E. coli and K. pneumoniae encoding the qnrA, qnrB, and qnrS enzymes genes in isolates from patients with leukemia in Tehran. Eighty E. coli and K. pneumoniae isolates were collected from patients with leukemia admitted to hospitals of Tehran. PCR technique was used to detect qnrA, qnrB, qnrS genes. The prevalence of qnrA, qnrB, and qnrS genes was 22.5, 26.75, and 46.25%, respectively. This study indicated that the high frequency of antibiotic resistance, qnrA, qnrB, and qnrS-producing E. coli and K. pneumoniae isolates, could be a major concern and further studies are needed for infection control measures in the patients affected by leukemia disease in Iran.