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There are no accurate data regarding the prevalence of Chlamydia trachomatis infection in Ukraine. This study aims to estimate the prevalence in the northeastern region of the country through reviewing nucleic acid amplification test results in patients of medical institutions in the Kharkov region during 2014–2016. Samples from 6920 patients (5028 women and 1892 men) aged 12–76 years were tested. The overall positivity rate was 4.5% (95% CI 4.0–5.0): 3.9% (95% CI 3.4–4.5) in women and 6.1% (95% CI 5.1–7.3) in men. The highest prevalence was found in the 16–20 (8.5%, CI 6.3–11.4) and 21–25 (8.0%, CI 6.7–9.4) year age groups. The prevalence in men was higher than in women in all investigated groups. The results show the need for more attention to the prevention, diagnosis, and treatment of chlamydial infection in these age groups of women and men in this region.