Abstract Introduction Many undergraduate students are newly sexually active adults with unsafe sexual practices. While sexual education curriculums exist, their understanding of reproductive and sexual health is unknown. Objective We sought to assess undergraduate students' knowledge and attitudes on reproductive and sexual health. Our aim was to identify variables associated with knowledge deficiencies. Methods A novel, cross-sectional questionnaire was created with local expert input. The survey assessed respondents’ demographics, attitudes, and knowledge of contraception/family planning, sexually transmitted infections (STIs), and infertility. The survey was distributed online from January to March 2021 to undergraduate students at two universities in Manitoba, Canada, through their students’ unions. Local ethics board approval was obtained, participation was voluntary, and data was anonymized. After calculating median and interquartile ranges according to the data distribution, categorical variables were analyzed with Chi-squared tests and continuous variables were analyzed with Kruskal-Wallis tests. Univariable and multivariable-adjusted logistic regression analysis were used to assess variables associated with knowledge differences. A p-value < 0.05 was considered statistically significant. Results There were 309 respondents out of approximately 28,816 possible undergraduate students (n=309, response rate 1.1%). Of the respondents, 239 (77.3%) were females, 146 (47.2%) were ≥22 years old, 49 (15.9%) were from a high socioeconomic status (SES), and 264 (85.4%) were from Manitoba. Few felt that their post-secondary education on contraception/family planning (26.2%), STIs (34.6%), and infertility (4.5%) was adequate. Many felt at risk of having an unplanned pregnancy (45.3%) and contracting a STI (34.6%). The overall mean knowledge score was 65.5%, with averages of 71.7% correct for STIs, 65.6% for infertility, and 59.2% for contraception/family planning. There were significant differences in knowledge scores with age (p = 0.012), location of high school completion (p = 0.004), and university attended (p=0.044). On multivariate analysis, higher knowledge scores were associated with identifying as female (male OR = 0.39, 95% confidence interval (CI): 0.18-0.87; p = 0.021), older age (age 21 OR = 6.04, 95% CI: 1.78-20.49; p = 0.004 and ≥22 years OR = 4.07, 95% CI: 1.35-12.26; p = 0.012), and university attended (University of Manitoba OR = 1.97, 95% CI: 1.11 – 3.49; p = 0.021). On subgroup analysis, family SES was associated with higher STI knowledge scores (OR = 2.19, 95% CI: 1.07 – 4.48; p = 0.032). Conclusions Undergraduate students face unique barriers in obtaining adequate reproductive and sexual health knowledge. In particular, younger males who attend smaller universities, and possibly those who are from lower SES, may be at greater risk of negative outcomes. We highlight the need for improved access to education and supports to assist undergraduate students in making more informed decisions. Disclosure No.