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Background With aging of the population, screening and prevention health programs for blood donors will increasingly be a priority. We aimed at: assessing the 10 year-cardiovascular disease (CVD) risk in blood donors, according to Italian CUORE risk score (CRS); determining the association of homocysteine (Hcy), lipoprotein (Lp)(a) and socio-demographic or lifestyle variables with estimated 10-year CVD risk. Methods Between June 2015 and July 2017, 1,447 (61.2% men) unselected blood donors (aged 18–69 years) were enrolled at the Blood Transfusion Service of the Pescara General Hospital, Italy. The project entailed evaluation of unalterable (age and gender) and modifiable CV risk factors (total cholesterol, HDL, LDL, triglycerides, fasting glucose, smoking, hypertension). The educational attainment, socio-demographic and lifestyle behavior information were obtained through a structured self-report questionnaire, and Health-related quality of life (HRQoL) through the Short Form Survey (SF-12). Plasma Hcy and Lp(a) were determined in the fasting state. Results A CRS within the moderate-high risk range was reported in 21.7% donors. Multivariate logistic regression, after adjustment for clinical and demographic variables, showed that Hcy [OR (95% CI): 1.09 (1.04–1.13); p < 0.001) and low educational attainment [1.71 (1.09–2.73); p = 0.019] are independent risk factors for moderate-to-high CVD risk. Instead, Lp(a), evaluated in 774 donors, was > 30 mg/dL in 22.4% of the examined population, but without any significant correlation with CRS. Conclusions Our study highlights a previously unappreciated need for CV risk assessment in blood donors, which may include evaluation of educational attainment as a non-traditional risk marker.