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This study aimed to assess patterns of sexual activity, preventive behaviors, contraceptive use, and the prevalence of chlamydia infection in student populations from two countries. 188 asymptomatic students of the University of Bielefeld, Germany and 590 students of the Navarra Public University, Spain were surveyed using a standardized self-administered questionnaire. Urine samples were analyzed by ligase chain reaction technique for chlamydia infection. Compared to German students, Spanish students were less likely to have more than one sex partner within the last 12 months (OR: 0.62; CI: 0.46–0.83), to have sexual intercourse more than one time per week (OR: 0.70; CI: 0.52–0.93) and to use oral contraceptives (OR: 0.16; CI: 0.10–0.26). They reported a higher use of condoms (OR: 2.93; CI: 2.01–4.27) and were more likely to always use condoms with a new sex partner (OR: 2.47; CI: 1.72–3.53). The prevalence of chlamydia infection was considerably higher in German students (4.8% in females; 2.2% in males) than in Spanish students, where no case was found. The higher frequency of sexual activity, a higher engagement in risk-taking sexual behavior and the lower use of barrier contraceptives may contribute to the higher prevalence of chlamydia infection in German students.