SUMMARYAim: This study investigated the cumulative incidence of Chlamydia trachomatis infection in women treated in gynaecology departments of healthcare facilities in two towns in Serbia and one town in the Former Yugoslav Republic (FYR) of Macedonia, including their medical records in public health reports.Methods: A cross-sectional observational research design with retrospective data collection during a five-year period (2008-2012) originated from women treated as in-and out-patients. The data included the results of cervical and urethral swab testing on Chlamydia trachomatis infection and women's gynaecological diagnoses in Pomoravlje County (the Institute of Public Health Cuprija "Pomoravlje" in Cuprija, Alba outpatient clinic, Paracin) and in Skopje (Clinic for Obstetrics and Gynaecology, "Mikrolab" laboratory).Results: The incidence of positive ELISA assay from samples from the Institute of Public Health "Pomoravlje" Cuprija and polyclinic Alba was 6.5% and 12.5%, respectively (p < 0.01). The incidence of positive DFA test from samples from the Clinic for Obstetrics and Gynaecology, Skopje and "Mikrolab" laboratory was 18.8% and 15.2%, respectively (p = 0.20). In Pomoravlje County and in Skopje 7.5% and 17.6% of urethral swab samples were positive for Chlamydia trachomatis, respectively (p < 0.01). The rate of microbiological samples tested for Chlamydia trachomatis in Pomoravlje County and Skopje was 48.4% and 21.2%, respectively (p < 0.01). One-year incidences of Infectio sexuales chlamydiales (A56) from 2007-2011 were significantly different among three data settings relating to Serbia, the FYR of Macedonia and Pomoravlje County (p < 0.001).Conclusions: The incidence of Chlamydia trachomatis positive cervical and urethral swabs in our study were highly variable between countries and within individual hospitals and caused by many factors.