BACKGROUND Chlamydia is the most prevalent sexually transmissible infection (STI) in Canadian adolescents. STI account for 20% of cases of infertility in Canada and 42% of ectopic pregnancies are attributable to previous chlamydia infection. Despite the importance of this infection, little is understood about young people's knowledge of it. METHODS A survey was conducted at a rural high school in Nova Scotia, Canada, to assess students' knowledge of chlamydia and associations of knowledge with gender and protective behaviours. Knowledge was assessed using true-false responses to 15 statements about chlamydia. Each statement was examined for differences in the percentage of correct responses by sex. Correct responses were summed, creating a knowledge score. Socioeconomic status variables and age were included in multivariate regression models to determine if they modified associations between knowledge score and protective behaviours seen in simple regression. RESULTS Eighty-six percent of registered students (n = 538) participated in the survey. Girls responded to 10 of the 15 knowledge statements significantly more often than boys. Respondents were least knowledgeable about their rights to confidential health services for chlamydia infection. Knowledge score was associated with use of both condoms and oral contraception at last intercourse in girls (odds ratio 1.15; 95% confidence interval 1.01-1.31). No association of knowledge score was seen with having had an STI test in the previous year. CONCLUSIONS School sexual health programs should make special efforts to meet the needs of male students, and programs and health professionals should include information about the confidential nature of sexual health services for adolescents.