As fluoroquinolone-resistant strains of Escherichia coli emerge, several risk factors for fluoroquinolone resistance have become evident, such as amino acid mutations in the quinolone resistance determining regions (QRDR) of gyrA and parC and previous use of fluoroquinolone. This study investigated risk factors for fluoroquinolone resistance and amino acid mutation in the QRDR in E. coli. We investigated the statistical correlation between each amino acid mutation and resistance to levofloxacin. We examined the minimum inhibitory concentration (MIC) of levofloxacin and the amino acid mutations of gyrA and parC by direct DNA sequence in E. coli clinically isolated from urinary tract infection (UTI) patients. We investigated risk factors for levofloxacin resistance, such as age, sex, and previous use of fluoroquinolone. We found a significant correlation between the number of mutations and resistance to levofloxacin (p < 0.001) and between the presence of underlying urinary tract disease and the presence of mutations (p = 0.004) by multivariate analyses. Three mutations in QRDR were demonstrated to be significantly correlated with levofloxacin resistance. In conclusion, these findings contribute to our understanding of the molecular mechanisms and risk factors for fluoroquinolone resistance.