OBJECTIVE Following a 9-year 60% decline, chlamydia positivity increased 46% from 1997 through 2004 among young sexually active women screened in Region X family planning clinics. The objective of this analysis was to systematically examine the influences of risk factors, changing laboratory test methods, and interclinic variability on chlamydia positivity during this period. METHODS We analyzed data from 520,512 chlamydia tests from women aged 15 to 24 years screened in 125 family planning clinics. Multivariate logistic regression modeling was used to adjust the annual risk of chlamydia for the demographic, clinical, and sexual risk behavior characteristics associated with infection and for the increasing use of more sensitive laboratory test methods. A generalized linear mixed model was used to adjust for interclinic variability. RESULTS We found a significant 5% annual increase in the risk of chlamydia even after adjusting for risk factors including laboratory test characteristics (odds ratio 1.05, 95% confidence interval: 1.04, 1.06). Variability among the clinics where screening occurred did not account for the increase. CONCLUSIONS Based on a review of all available data, we concluded that there was a true increase in chlamydia positivity over the 8-year period.