Objective: Sexually transmitted infections (STIs) disproportionately affect young people and can result in severe health consequences. Accurate knowledge about STIs can play an important role in STI prevention. Prior quantitative research has found that college students' knowledge about STIs is insufficient. However, there is a dearth of information regarding their specific STI knowledge deficiencies. This study sought to fill this gap by using a qualitative approach to elucidate gaps in students' STI knowledge. Design: Qualitative content analysis of responses to an open-ended online survey question. Setting: A large public Midwestern university in the USA. Method: Students (N = 289) watched a web-based STI health education programme covering basic information about Chlamydia, gonorrhoea, human papillomavirus (HPV), herpes and syphilis as a course requirement. Afterwards they answered an open-ended question about what information, if any, was new to them. Themes and sub-themes were identified, exemplar quotes were selected for illustrative purposes, and frequencies were calculated. Results: Ten overarching themes were identified about what students learned: everything/a lot (19.7%), prevention (16.6%), treatment/cures (15.6%), prevalence/statistics (14.9%), nothing/not much (14.2%), symptomatology (12.8%), everything about a specific STI (9.7%), health consequences (6.6%), transmission (5.5%) and testing (2.4%). Participants reported learning about HPV most frequently compared to other STIs. Conclusion: Findings demonstrate critical gaps in college students' STI knowledge and provide a deeper understanding of specific knowledge deficiencies. Study findings highlight important sexual health content that should be integrated into health education initiatives in academic, community and other settings, and provides recommendations on how to do so.