Background There is a paucity of population-based data on chlamydia in pregnancy despite rising rates in US women. Our objectives were to assess chlamydia prevalence by age group and to identify factors associated with infection in pregnant women to inform screening guidelines. Methods This cross-sectional study included pregnant women tested for chlamydia who delivered at the University of Alabama at Birmingham between November 1, 2012, and December 31, 2017. The primary outcome was chlamydia prevalence, defined as a positive urogenital chlamydia nucleic acid amplification test result documented in the electronic medical record. Multivariable logistic regression was used to identify factors associated with infection. Results Among 17,796 women who delivered during the study period, 13,657 (77%) had chlamydia testing performed at the University of Alabama at Birmingham. Chlamydia prevalence (95% confidence interval) was 7.4% (7.0%-7.9%). Age-stratified prevalence rates were 14.6%, 4.3%, and 1.7% for women younger than 25 years, 25 to 29 years, and 30 years or older, respectively. Chlamydia in pregnancy remained strongly associated with age (adjusted odds ratio [95% confidence interval], 7.2 [5.6-9.2] for age <25 years, and 2.3 [1.7-3.0] for ages 25-29 years, when compared with >30 years) after adjustment for race, urban residence, and insurance status. Conclusions Among pregnant women living in the southeastern United States, chlamydia was detected in 1 of 14 women who were tested. Chlamydia positivity was highest among women younger than 30 years. Study findings support broad screening for chlamydia in pregnancy.