Ureaplasmaspp. cause several disorders, such as nongonococcal urethritis, miscarriage, and preterm delivery with lung infections in neonates, characterized by pathological chorioamnionitis in the placenta. Although reports on antibiotic resistance in Ureaplasmaare on the rise, reports on quinolone-resistant Ureaplasmainfections in Japan are limited. The purpose of this study was to determine susceptibilities to five quinolones of Ureaplasma urealyticumand Ureaplasma parvumisolated from perinatal samples in Japan and to characterize the quinolone resistance-determining regions in the gyrA, gyrB, parC, and parEgenes. Out of 28 clinical Ureaplasmastrains, we isolated 9 with high MICs of quinolones and found a single parCgene mutation, resulting in the change S83L. Among 158 samples, the ParC S83L mutation was found in 37 samples (23.4%), including 1 sample harboring a ParC S83L–GyrB P462S double mutant. Novel mutations of ureaplasmal ParC (S83W and S84P) were independently found in one of the samples. Homology modeling of the ParC S83W mutant suggested steric hindrance of the quinolone-binding pocket (QBP), and de novoprediction of peptide structures revealed that the ParC S84P may break/kink the formation of the α4 helix in the QBP. Further investigations are required to unravel the extent and mechanism of antibiotic resistance of Ureaplasmaspp. in Japan.