The low virulence of quinolone- and fluoroquinolone-resistant Escherichia coli strains is known, although the reasons for this remain unclear. We surveyed the mutation patterns of quinolone resistance determining regions (QRDRs), phylogenetic distribution, prevalence of 18 urovirulence genes, and PAIusp subtypes in 89 fluoroquinolone-resistant E. coli (FQREC) isolates obtained from patients with cystitis and compared them with those of their fluoroquinolone-susceptible counterparts (FQSEC). Phylogenetic group B2 was significantly less prevalent in FQREC than in FQSEC (49% versus 78%; P = 0.0138), but it still dominated, followed by phylogroup D (35%), in FQREC. When the prevalences of virulence factor (VF) genes were compared between FQREC and FQSEC, sfa/foc, cnf1, hly, kpsMT, ompT, ibeA, usp, and iroN showed significantly lower prevalences in FQREC than in FQSEC (1.1% versus 24% [P < 0.0001], 0% versus 29% [P < 0.0001], 7.9% versus 33% [P < 0.0001], 74% versus 90% [P = 0.01], 71% versus 87% [P = 0.017], 5.6% versus 37% [P < 0.0001], 54% versus 82% [P < 0.0001], and 7.9% versus 32% [P = 0.0001], respectively), whereas aer, iha, and ETTT showed significantly higher prevalences in FQREC (85% versus 36% [P < 0.0001], 66% versus 29% [P < 0.0001], and 53% versus 16% [P < 0.0001], respectively). Furthermore, a similar difference in prevalences of uropathogenic VF genes was seen between FQREC and FQSEC in phylogroup B2. This indicated that the low virulence in FQREC was intimately correlated with a lesser distribution of VFs in phylogroup B2, which dominated in FQREC and FQSEC. It was interesting that the mutation pattern of Ser83Leu and Asp87Asn encoded in gyrA and Ser80Ile and Glu84Val encoded in parC was frequently found in FQREC isolates that belonged to phylogroup B2 and that most of these isolates showed PAIusp subtype 2a. PAIusp subtypes 1a, 1b, and 2b, which were frequently seen in FQSEC, were rarely found in FQREC. These results suggested that the acquisition of fluoroquinolone resistance, e.g., mutations in QRDRs, might be a specific event in limited strains, such as those that possess PAIusp subtype 2a in phylogroup B2.