Objectives Recently, we reported a high prevalence (16%) of urogenital Chlamydia trachomatis infections among women in a rural setting in South Africa. Molecular epidemiological studies on C. trachomatis infections could provide insights into the characteristics of this epidemic, yet such data are not available. The objective of this study was therefore to assess the distribution of C. trachomatis strains among women from a South African rural community, the Mopani district, and to compare it with strains from Amsterdam, the Netherlands. Methods High-resolution multilocus sequence typing (hr-MLST) was used to study urogenital C. trachomatis infections in women visiting primary healthcare facilities across rural Mopani District in Limpopo Province, South Africa. Sequence types (STs) were compared with 100 strains from women visiting the sexually transmitted infection clinic in Amsterdam, the Netherlands. Results Full hr-MLST data were obtained for C. trachomatis infection in 43 women from Mopani district. Using the complete hr-MLST profile of all 43 women from Mopani district, 26 STs could be identified, of which 18 (69%) were novel to the hr-MLST database. The remaining STs clustered together with strains from Amsterdam. Conclusions Hr-MLST data revealed a diverse molecular epidemiology with novel STs and a specific cluster for the Mopani district. Also C. trachomatis types that occur worldwide were detected.