Abstract Background Chlamydia trachomatis (chlamydia) is the most diagnosed sexually transmitted infection in Belgium. Screening programs focus on young women, due to the implications of chronic asymptomatic infections for reproductive health. Thereby, the frequency of infections in men and older adults is underestimated. This study aimed to estimate the point-prevalence of chlamydia in the broader Belgian population, to inform evidence-based prevention and control strategies. Methods We conducted two cross-sectional prevalence studies of chlamydia infection in the population of Belgium aged 16–59 years, 2018–2020. In the CT1 study 12,000 representative individuals were randomly selected from the national register and invited by letter to collect a urine sample at home. The CT2 study used urine samples collected through the Belgian Health Examination Survey. Molecular detection of chlamydia DNA was performed using Xpert® or Abbott Real-Time CT/NG assays. Weighted estimated prevalence and 95% confidence interval (CI) was calculated per gender and age groups of 16/18–29, 30–44 and 45–59 years, relative to the general Belgian population. Data collected on sociodemographic variables and sexual behavior were used to identify potential risk factors for chlamydia infection through calculation of the odds ratio (OR). Results The population-wide weighted estimated prevalence was 1.54% (95% CI 0.78–3) in CT1 and 1.76% (95% CI 0.63–4) in CT2. We observed no statistically significant difference between men and women or age groups. Civil relationship status (OR = 14.1 (95% CI 1.78–112), p < 0.01), sexual intercourse with a casual partner (OR = 6.31 (95% CI 1.66–24.1), p < 0.01) and > 3 sexual partners in the last 12 months (OR = 4.53 (95% CI 1.10–18.6), p = 0.02) were associated with higher relative risk for chlamydia infection. Conclusion Nationwide prevalence studies are relevant to assess the distribution of chlamydia and inform public health actions. The overall low prevalence and heterogeneous distribution of ...