Objective: Epstein-Barr virus (EBV) is the cause of infectious mononucleosis, which disproportionately affects university students. This population has the potential to benefit from a prophylactic EBV vaccine trial. Our objectives were to determine EBV infection status and associated demographic/lifestyle factors among first year undergraduate university students at the beginning and end of first year. Methods EBV infection status was assessed by testing for circulating IgG class antibodies against EBV viral capsid antigen. Results Of 198 starting students; 56.1% were positive for EBV antibodies with a higher rate in women (64.8%) than male (41.1%); p = 0.002. A history of deep kissing was associated with a higher rate of EBV antibody positivity. On follow-up 8 months later at the end of freshman year, 22.4% had acquired EBV antibodies for a primary infection incidence of 33.6/100 person years. Conclusion These findings indicate that our first year undergraduate population contains sufficient EBV-naïve subjects for a prophylactic vaccine trial.