Background: Previous studies demonstrated high levels of Chlamydia trachomatis (CT) infections within American Indian/Alaskan Native (AI/AN) populations but there are few analyses of CT prevalence in these populations over time. Methods: We analyzed data from 7374 visits at which diagnostic tests for CT were collected in AI/AN women aged 15 to 24 years seen at family planning clinics associated with the Region X Infertility Prevention Project. Trends in population characteristics and test pos- itivity were examined and compared with non-AI/AN women tested in the same setting and time period. Chlamydia positivity was adjusted for changes in diagnostic test type. Multivariable logistic regression was used to identify characteristics associated with infection. Results: Adjusted CT positivity in AI/AN women rose from 7.8% to 11.0%, which was 1.5 to 2.2 times the non-AI/AN population levels over the study period (absolute difference 2.8%-6.6%). Differences persisted after correction for test type and age. Temporal changes in positivity among AI/AN women were associated with a rise in reported risk behaviors and decline in age of the population being tested. Risk factors associated with positivity among AI/AN women were younger age, ≥1 behavioral risks, ≥1 clinical findings, partner with chlamydia, chlamydia in past year, and pregnancy related visit. Conclusions: AI/AN women had consistently higher levels of chla- mydia positivity than non-Native women, even after adjustment for age and diagnostic test. Further investigation of risks for chlamydia, related outcomes, access to screening, sexual networks, and enhanced surveillance would be beneficial for improving health in this vulnerable population. Copyright © 2008, American Sexually Transmitted Diseases Association.