Moxifloxacin (MF) - resistant mutants of Mycoplasma hominis (M. hominis) were generated by stepwise selection in increasing concentrations of MF, and six strains of MF resistant M. hominis mutants - M1, M4, M8, M16, M32, and M64 - in which MICs of MF were 0.5, 4, 8, 16, 32, 64 ${mu}g$/ml, respectively, were generated. Compared to the sequence of M. hominis PG21, all mutants harbored amino acid substitutions of Arg-163 Thr in GyrA, and Pro-445 Gln in ParE. While the concentrations were getting higher, an additional amino acid substitution was found at Ser-153 Lys in GyrA (${geq}4{mu}g/ml$), Ser-91 Ile in ParC (${geq}16{mu}g/ml$), and Val-450 Phe (${geq}64{mu}g/ml$) in GyrB. These substitutions seem to have an impact on resistance to MF, and GyrB change was found only in the highest concentration and seems to be associated with high-level resistance to MF. This, as far as we know, is the first description of a relationship between MF resistance phenotype and genotype.