Abstract： Objective To test the in vitro susceptibility to macrolides of urogenital Chlamydia trachomatis (Ct) isolates, screen resistant Ct strains, and to explore resistance mechanism at the molecular level. Methods A total of 42 Ct strains were isolated from cervical or urethral swab samples and propagated in McCoy cells until the infection rate reached more than 90%. Then, susceptibility test was performed to evaluate the activity of three macrolides. Reverse transcription PCR and PCR were used to amplify two macrolide-resistance related genes, i.e., 23S rRNA gene and L4 gene, respectively in 2 erythromycin-resistant Ct strains and 4 erythromycin-sensitive strains followed by direct sequencing. Results The minimal inhibitory concentration (MIC) varied from 0.5 to 2 mg/L for erythromycin, 0.008 to 0.032 mg/L for clarithromycin, 0.125 to 0.5 mg/Lfor azithromycin. Erythromycin resistance was found in 2 isolates with the MIC value being 2 mg/L. Two mutations, C2452A and T2611A/C (Escherichia coli numbering) in the 23S rRNA gene, were detected in the resistant strains only, while the other 2 mutations, Pro113Leu and Pro156 Ala in L4 gene, were observed in all the tested strains. Conclusions Erythromycin-resistant Ct strains have emerged in clinical settings. The low-level erythromycin resistance may be associated with C2452A and T261 1C mutations in the 23S rRNA gene, whereas the point mutations in L4 gene is unlikely related to erythromycin resistance.