Background: The Internet is widely used by adolescents for sexual health information and bears the potential to increase knowledge and positively affect behavior. Objective: The objective of this study is to assess students’ preferences when looking for sexual health information online. Methods: We conducted a cross-sectional survey among ninth grade students in a convenience sample of 13 secondary schools in Berlin, Germany. During a regular school period, participants were requested to rate the importance they attribute to nine aspects of sexual health websites in a paper-based questionnaire. Bivariate and multivariable analyses were used to assess awareness and preferences by gender, age, migrant background, and school type. Results: Of 1190 eligible students, 1177 (98.91%) students with a mean age of 14.6 (SD 0.7) years participated, 52.52% (605/1152) were male, and 52.94% (612/1156) had at least one parent born abroad. Participant numbers were spread equally across three types of secondary schools in Berlin. Website aspects most frequently cited as important were easily comprehensible wording (88.33%, 961/1088), clear information layout (80.57%, 871/1081), and reliability of the website’s publisher (79.28%, 857/1081), whereas the visual style of a website was deemed important by the lowest number of students (35.13%, 378/1076). There was a marked gender difference in the importance students attached to website publisher reliability. Although 437/515 (84.9%) of female participants regarded this as important, only 420/566 (74.2%) of male participants did likewise (P<.001). In multivariable analyses, demographic differences were also particularly visible in the importance of publisher reliability: male participants were significantly less likely to find this aspect important (OR 0.50, 95% CI 0.37-0.69). The odds ratio for students with migrant background was 0.64 (95% CI 0.50-0.81, reference=no migrant background) and OR 2.04 (95% CI 1.03-4.03) for students in the most academic school type (reference=least academic). Conclusions: Students prefer easily understandable online resources. Setting up sexual health websites according to the explicit preferences of the target audience might encourage usage, especially by those subpopulations less likely to critically assess information validity: male adolescents, children of immigrants, and the academically disadvantaged.