This is an observational multicentric cross-sectional study aiming at assessing the association between ABO blood groups and SARS-CoV-2 seroprevalence among the blood donors in Puglia region. Data on ABO and Rh blood groups and demographic characteristics were obtained from Blood Bank Information System. All donors were screened for SARS-CoV-2 IgG antibodies. Comparison of seroprevalence among blood groups and the association between the recorded variables and seroprevalence were evaluated. A total of 35,709 donors from 22 centers were included, with a seroprevalence of 6.8%. The distribution of ABO phenotypes was blood type O (46.8%), A (34.0%), B (14.7%), and AB (4.5%). Among the 2416 donors reactive for SARS-CoV-2 IgG, the prevalent phenotype was blood type O (43.1%), followed by A (37.7%), B (14.2%), and AB (5%). The seroprevalence of phenotype A and AB was 7.5%, followed by B (6.5%) and O (6.2%). According to the adjusted analysis, there was an increase in seroprevalence in groups A and AB, compared to group O, and an increase in males compared to females. A possible effect modification was observed after stratifying for sex (p = 0.0515). A significantly lower prevalence of blood type O was found compared to A and AB, whereas no association was observed between Rh factor and seroprevalence. We hypothesized that the A antigen present in blood type A and AB can play a role in the binding of SARS-CoV-2 to ACE2 receptors, resulting in an increased risk of infection. Furthermore, natural anti-A/anti-B antibodies produced in group O could block viral adhesion to cells and explain a lower risk of infection.