OBJECTIVES: To determine the prevalence and risk factors of genital Chlamydia trachomatis (CT) among school-going sexually experienced male and female adolescents in Panama. METHODS: We conducted two multisite cross-sectional studies using two-stage cluster sampling to select adolescents aged 14-19 years attending urban public high schools (URB) in Panama City, San Miguelito, Colón and Panama Oeste from 2015 to 2018, and in the rural Indigenous Comarca Ngäbe-Buglé (CNB) from July-November 2018. CT testing was performed by real-time PCR on urine samples. Random-effects logistic regression accounting for sample clustering was used to identify risk factors. RESULTS: We enrolled 3166 participants (54.3% females), median age 17 years (IQR: 15.9-18.1), with no difference by sex. Sexual experience was reported by 1954 (61.7%) participants. Combined CT prevalence was 15.8% (95% CI: 14.2 to 17.4), with no significant differences by region (URB=16.5%, 95% CI: 14.7% to 18.6%; CNB=13.6%, 95% CI: 10.9% to 16.8%; p=0.12). In an age-and-region-adjusted analysis, CT prevalence was higher among female participants compared with males (21.6% vs 9.1%, adjusted OR (AOR)=2.87, 95% CI: 1.62 to 5.10). Among sexually experienced females, CT prevalence was higher among those who reported ≥3 lifetime sex partners compared with one partner (33.5% vs 15.3%, AOR=2.20, 95% CI: 1.09 to 4.07); and among those reporting at least one pregnancy compared with nulligravidae participants (30.9% vs 13.8%, AOR=1.89, 95% CI: 1.05 to 3.43). In unadjusted analyses among males, CT was associated with older age (11.5% among those aged 18-19 years vs 3.4% among those aged 14-15 years, OR=3.69, 95% CI: 1.10 to 12.33). CONCLUSIONS: We report high CT prevalence among sexually experienced, school-going adolescents in Panama. Female adolescents, particularly those with multiple sex partners and a history of pregnancy, were at highest risk. Adolescent-targeted CT screening should be implemented in Panama. Additionally, evidence-based comprehensive sexuality education will be imperative.