BACKGROUND: Young people aged 15-24 years continue to be disproportionately affected by HIV globally. In sub-Saharan Africa, utilization of HIV prevention services remains suboptimal. Education plays a pivotal role in enhancing HIV prevention knowledge among youth, but its effectiveness is influenced by factors such as age, gender, and socioeconomic status. The objective of this study was to investigate how education status, age, and gender influence awareness use, and preferences of HIV prevention methods among youth in rural southwestern Uganda. METHODS: A cross-sectional quantitative study was conducted in March 2022 among 216 adolescents and young adults aged 15-24 years in Rubirizi District, southwestern Uganda. Participants were recruited using stratified random sampling from three publicly funded schools and snowball sampling from local trading centers to include both school-going and non-school-going youth. Data were collected using a structured questionnaire translated into the local language. Descriptive statistics and inferential analyses were performed using R software, with statistical significance set at p < 0.05. RESULTS: A majority (72%) perceived themselves at low risk of HIV infection, with school-going youth demonstrated higher awareness of abstinence (75% vs. 32%, < 0.001) and prevention of mother-to-child transmission (PMTCT) (46% vs. 25%, = 0.002) compared to non-school-going youth. Younger individuals were more likely to practice abstinence (mean age 18.9 years vs. 19.7 years, = 0.017). Males also had higher awareness of being faithful (59% vs. 44%, p = 0.04). Private doctors (46%) and hospitals (47%) were the most favored access points for PrEP. Younger participants expressed greater concern about daily pill-taking inconvenience (mean age 20.4 years vs. 19.2 years, p = 0.04). Radio (73%) and television (41%) were the most preferred methods for HIV information dissemination. Females preferred magazines more than males (21% vs. 10.3%, p = 0.046). CONCLUSION: Education status, age, and gender influence HIV prevention awareness through differences in access to information, risk perception, and preference of prevention methods among youth in rural Uganda. Therefore, aligning HIV prevention strategies with the specific needs and preferences of different youth subgroups can enhance awareness, risk perception, and utilization of HIV prevention methods.