Objective To survey the distribution of quinolonesresistant genes, DNA gyrase, multiple-drug efflux pump and aminoglycoside modification enzyme (AME), in the clinical isolates of Escherichia coli. Methods Sixty strains of E.coli isolated from hospitalized patients were collected from January of 2006 to October of 2008. Susceptibility tests for 16 antibiotics were carried out with disk diffusion method. Six quinoloneresistant genes ( gyrA, qnrA, qnrB, qnrS, qepA, mdfA) and a AME aac(6′)-Ⅰ b were detected by PCR and sequencing. Results Among 60 isolates, resistant rates of ciprofloxacin and levofloxacin were both 78.4% (47/60). gyrA mutation was found in 55 resistant isolates. All mutated genes were sequenced and blasted with GeneBank of NCBI, and 3 new gyrA mutants were discovered. All resistant strains were mdfA genepositive and 9 isolates were aac(6′)-Ⅰ b positive by PCR assay in which 5 stains carried bifunction gene aac(6′)-Ⅰ b-Cr by sequencing identification. Conclusions Mutation of gyrA gene may be the main mechanism of E.coli. resistant to quinolones. The quinoloneresistant genes aac(6′）-Ⅰ b-Cr and new mutants of gyrA were discovered from resistant isolates of E.coli. The resistant gene mdfA of E.coli is firstly reported in Chinese mainland.