Background: Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) infections are common, causing significant morbidity in pregnancy (congenital CMV) and transplant recipients (CMV, EBV). Canadian prevalence data are needed to model disease burden and develop strategies for future vaccines. We estimated prevalence using screening data from blood donors and solid organ transplant (SOT) donors and recipients. Methods: We retrospectively analyzed CMV and EBV serology from Alberta SOT donors ( n = 3,016) and recipients ( n = 4,614) (1984–2013) and Canadian Blood Services blood donors ( n = 1,253,350) (2005–2014), studying associations with age, sex, organ, year, and geographic region. Results: CMV seroprevalence rises gradually with age. By age 70, CMV seropositivity ranged from 67% (blood donors) to 73% (SOT recipients). Significant proportions of women of child-bearing age were CMV-seronegative (organ donors, 44%; SOT recipients, 43%; blood donors, 61%). Blood donor CMV seroprevalence decreased from 48% in Western Canada to 30% in Eastern Canada. Women were more likely to be CMV-seropositive (ORs = 1.58, 1.45, and 1.11 for organ donors, SOT recipients, and blood donors, respectively) and EBV-seropositive (ORs = 1.87 and 1.46 for organ donors and SOT recipients, respectively). EBV prevalence rises rapidly, and by age 17–29 years, 81% of SOT recipients and 90% of organ donors were seropositive. Conclusions: Canada has relatively low and perhaps decreasing age-specific EBV and CMV prevalence, making Canadians vulnerable to primary infection-associated morbidity and suggesting benefit from future vaccines. Collection and analysis of routine serology screening data are useful for observing trends.