Objectives: There is only limited information on the antimicrobial susceptibilities and resistance genes of Ureaplasma parvum in South Africa. This study was designed to detect and characterize resistance genes in U. parvum. Methods: Fifteen U. parvum isolates were investigated employing the broth microdilution method (tetracycline, doxycycline, ofloxacin, erythromycin, azithromycin and josamycin). Gene analyses were performed on target regions of: tet(M); gyrA, gyrB, parC and parE; erm(A), erm(B), erm(C) and erm(E); msr(A), msr(B), msr(C) and msr(D); 23S rRNA operons; and L4 and L22 ribosomal proteins. Results: Seven of the U. parvum isolates were fully susceptible to the antibiotics tested. Five strains exhibited resistance to tetracycline (MICs 16-256 mg/L), one strain was resistant to ofloxacin (MIC 128 mg/L) and four strains were resistant to macrolides (MICs 128 mg/L); two strains showed dual resistance to tetracycline and erythromycin. The five tetracycline-resistant strains were found to have mosaic tet(M) genes, with one strain containing different specific regions to those previously described. Mutations in the L22 ribosomal protein were seen in three strains that were resistant to erythromycin (two strains) and erythromycin + azithromycin (one strain). For a further strain that was resistant to erythromycin and azithromycin, possible mechanisms of resistance remained elusive. Conclusions: This is the first report of quinolone, erythromycin and azithromycin resistance development in U. parvum from South Africa. A point mutation in parC (Pro-57 → Leu) and two novel mutations in parE (Ile-73 → Thr and a methionine insertion at codon 86) were found in an ofloxacin-resistant strain. The study reinforces the adaptability of U. parvum to develop resistance and acquire, modify and maintain transposon-located resistance genes.