One of the treatment options of Escherichia coli and Klebsiella spp. infections which are the most common opportunistic pathogens of gram-negative sepsis is quinolones. Resistance to quinolones which act by disrupting DNA synthesis has been increasing. Horizontal transfer of plasmid-mediated quinolone resistance (PMQR) genes play an important role in the spread of resistance. The data about the prevalence of PMQR genes in our country is quite limited. The aim of this study was to investigate the presence of known PMQR genes namely qnrA, qnrB, qnrC, qnrS, qnrD, aac(6′)-lb-cr, qepA and oqxAB amongst quinolone-resistant E coli and Klebsiella spp. strains isolated from blood cultures. One hundred twenty seven E.coli and 66 Klebsiella isolates detected as nalidixic acid- and/or ciprofloxacin-resistant by phenotypical methods, from 193 blood samples of 187 patients admitted to Karadeniz Technical University, Faculty of Medicine, Department of Medical Microbiology, Bacteriology Unit of Patient Service Laboratory between January 2012 to August 2013 were included in the study. The presence of PMQR genes were investigated by polymerase chain reaction (PCR) and for the detection of aac(6′)-lb-cr variants PCR-restriction fragment length polymorphism (PCR-RFLP) method was used. The positive bands were sequenced using the same primers, and aligned with formerly defined resistance gene sequences, and confirmed. In the study, 56.7% (72/127) of Eco//and 19.7% (13/66) of Klebsiella spp. isolates, with a total of 44% (85/193) of all the isolates were found to be phenotypically resistant to quinolones. Of the 13 resistant Klebsiella isolates, 11 were K.pneumoniae, and two were K.oxytoca. Extended-spectrum beta-lactamase (ESBL)-producing isolates showed higher resistance (50/80, 62.5%) to quinolones than the negative ones (35/113, 30.9%). The prevalence of quinolone resistance genes among resistant E coli and Klebsiella spp. isolates was determined as qnrA, 1.4% and 15.4%; qnrB, 4.2% and 61.5%; qnrS, 1.4% and 7.7%; qepA, 1.4% (only E.coli); aac(6′)-1b-cr, 38.9% and 92.3%; and oqxAB, 1.4% and 84.6%, respectively. qnrC and qnrD genes were not detected. Carriage of multiple resistance genes were observed more frequently among resistant Klebsiella isolates than E.coli strains. As a result, in this study investigating the contribution of transferable genes to quinolone resistance, prevalence of PMQR genes in quinolone-resistant and blood isolates of E.coli and Klebsiella in our university hospital serving the region were found to be higher than the current data reported from the other studies in our country. Furthermore, this study presented the initial data for the first time in our country on the prevalence of qnrD which was undetected, and the frequency of oqxAB gene in clinical samples. However, location of oqxAB gene needs to be confirmed by conjugation or hybridization methods.