Objectives: We compare data collected by England's National Chlamydia Screening Programme (NCSP) with national probability survey data to examine demographic and behavioural differences that may be important in understanding who the NCSP is reaching and interpreting chlamydia positivity. Methods: Data for 538 119 men and women aged 16-24 years who were screened in 2008 and data collected from 2180 interviewees in Britain's second National Survey of Sexual Attitudes and Lifestyles 1999-2001 (Natsal-2), of whom 644 were tested for chlamydia, were compared using the χ2 statistic and logistic regression. Results: Compared with Natsal-2, the NCSP tested more women (67% vs 49%). NCSP participants were more likely to be younger: 29% were 16-17 years versus 16% of men and 15% of women in Natsal-2; from ethnic minority groups: 17% of men and 14% of women versus 8% and 6%, respectively, in Natsal-2; not to have used condoms at last sex: 66% of men and 68% of women versus 48% and 63%, respectively, in Natsal-2: and more likely to report two or more partners in the last year: 62% of men and 47% of women versus 47% and 30%, respectively, in Natsal-2. In multivariate analyses, higher AOR of chlamydia positivity were found for those reporting non-use of condoms and for those reporting multiple partners in both the NCSP and Natsal-2. Conclusions: The NCSP is testing young people at increased risk of chlamydia. The impact of this testing bias on the effectiveness of the programme should be evaluated.