Objectives: Chlamydia trachomatis, which is asymptomatic in most women, causes significant adverse effects for pregnant women and neonates. No programmes conduct antenatal screening in Latin America. We determined chlamydia prevalence, feasibility and acceptability of chlamydia screening, and adherence to treatment in pregnant women in two urban public hospitals in Lima, Peru. Methods: We offered chlamydia screening using self-collected vaginal swabs to pregnant women ≥16 years of age during their first antenatal visit. Chlamydia-infected women were contacted within 14 days and asked to bring partners for counselling and directly observed therapy with oral azithromycin. Unaccompanied women received counselling, directly observed therapy, and azithromycin to take to partners. Test of cure was performed ≥3 weeks after treatment. Results: We approached 640 women for the study and enrolled 600 (93.8%). Median age was 27.3 years (range 16-47), median lifetime partners 2.3 (range 1-50), and median gestational age 26.1 weeks (range 4-41). Chlamydia prevalence was 10% (95% CI 7.7% to 12.7%). Of 60 infected patients, 59 (98%) were treated with one dose of azithromycin. Fifty-two of 59 (88%) returned for test of cure, all of whom were treated successfully, with 46 (86%) achieving negative test of cure with one dose of azithromycin, and 6 (12%) after retreatment with a second dose. Conclusions: C. trachomatis screening and treatment in pregnancy was feasible and highly acceptable in two urban hospitals in Peru. Chlamydia prevalence was high. Clinical trials to evaluate efficacy and cost-effectiveness of chlamydia screening, and treatment of pregnant women to prevent adverse pregnancy outcomes in low-resource settings, are warranted.