Quinolones are used extensively to treat Shigella flexneri infection, and plasmid-mediated quinolone resistance (PMQR) determinants were recently reported. A total of 26 S. flexneri isolates collected from Anhui province, China, in 2005 were screened for PMQR determinants, bla gene, gyrA and parC genes, by PCR and sequencing. PMQR determinants were present in 53.8% (14 of 26) of isolates and qnrA(1), qnrS(1), qnrS(2), aac(6')-Ib-cr and qepA were present in 30.8, 11.5, 3.8, 19.2 and 11.5% of those isolates, respectively. All PMQR determinants' positive isolates exhibiting 8 different genetic clones had mutations in gyrA and parC genes, and 11 carried bla genes, including 7 bla(CTX-M) with resistance to cefotaxime. These isolates were resistant to nalidixic acid and 57.1% (8 of 14) of them were resistant to ciprofloxacin and levofloxacin. Our data show that there was a high prevalence of PMQR determinants in S. flexneri isolates from China.