BACKGROUND: The purpose of this study was to examine variation in positivity within the English National Chlamydia Screening Programme during 2007/2008. METHODS: Data were analyzed using multivariable logistic regression. The outcome measure was positivity. Funnel plots were used to explore variation in positivity according to screening volume. RESULTS: Three hundred and thirty-four thousand nine hundred and two screening tests were done, 29% of which were in men. Overall positivity was 7.6% in men and 9.3% in women. For men, positivity increased rapidly to plateau from ages 19 to 24. For women, rates peaked at 18 years-those aged 21 being at the same risk of chlamydial infection as 16-year-olds. For men and women, positivity was generally higher for those of black or mixed ethnicity compared with whites, whereas Asians were at lower risk. Similarly, risk of infection for men and women varied by screening venue. Multivariable analysis showed that, for men and women positivity varied significantly with age, ethnicity, screening venue attended, whether the young people had had a new sexual partner in the past 3 months, and whether the patient had had 2 or more sexual partners in the past year. Positivity did not vary significantly with implementation phase. CONCLUSIONS: This is the largest description of testing for Chlamydia trachomatis in healthcare and nonhealthcare settings outside Genitourinary Medicine clinics in England and allowed a detailed analysis of positivity by age and ethnic group. Considerable heterogeneity exists and local health service commissioners need to ensure that the implementation of chlamydial screening reflects these differences.