Background: Following a small pilot study in 2003, a study was set up to determine the prevalence of genital Chlamydia trachomatis infection in young women presenting to Family Planning NSW centres across New South Wales and to evaluate the characteristics of those infected. Methods: A cross-sectional survey of 621 consecutive women aged from 16 to 24 years was carried out over a 3-month period in 2004 at five Family Planning NSW centres. Urine samples were tested for C. trachomatis using the polymerase chain reaction (PCR) method. Women with a positive result were reviewed and treated. Results: Of 925 eligible clients, 621 (67%) were recruited to the study. Chlamydia trachomatis was detected in 35 of the 621 recruits (5.6%, 95% CI 3.8–7.4). The prevalence at the Hunter centre was significantly higher than the combined prevalence at the other four participating centres (9.7% compared with 3.9%; P = 0.008). Two characteristics were identified as independent predictors of infection in this study: ‘reporting a recent change of partner in the past three months’ (crude odds ratio (OR) 3.33, 95% CI 1.67–6.64) and ‘reporting three or more partners in the past year’ (crude OR 3.69, 95% CI 1.83–7.46). Reported condom use, a history of one or more sexually transmissible infections and current combined oral contraceptive pill use were not associated with infection in this study. Conclusions: The prevalence of C. trachomatis infection is sufficiently high to support targeted testing of 16–24-year-old women in the Family Planning NSW setting and informs the development of a national screening strategy.