Quinolone susceptibility was analyzed in 17 clinical isolates of Corynebacterium striatum and 9 strains of Corynebacterium amycolatum by the E-test method in Mueller-Hinton agar plates. The C. striatum ATCC 6940 strain was used as a control strain. The amplified quinolone resistance determining regions of the gyrA genes of C. amycolatum and C. striatum were characterized. Four in vitro quinolone-resistant mutants of C. amycolatum were selected and analyzed. Both in vivo and in vitro quinolone-resistant strains of C. amycolatum showed high levels of fluoroquinolone resistance in strains with a double mutation leading to an amino acid change in positions 87 and 91 or positions 87 and 88 (unusual mutation) of GyrA, whereas the same concomitant mutations at amino acid positions 87 and 91 in GyrA of C. striatum produced high levels of resistance to ciprofloxacin and levofloxacin but only showed a moderate increase in the MIC of moxifloxacin, suggesting that other mechanism(s) of quinolone resistance could be involved in moxifloxacin resistance in C. amycolatum. Moreover, a PCR-RFLP-NcoI of the gyrA gene was developed to distinguish between C. amycolatum and C. striatum species.