Coxsackieviruses are human enteroviruses, which have been associated with myocarditis/pericarditis and sudden death. In one investigation (Spanakis N, Manolis EN, Tsakris A, Tsiodras S, Panagiotopoulos T, Saroglou G, and Legakis NJ: J Clin Pathol 2005;58:357–360), a cluster of cases of fatal myocarditis in Greece was linked to coxsackievirus B3. The information from this investigation prompted us to study serologically the prevalence of coxsackieviruses B throughout Greece. Sera were obtained from 506 healthy blood donors from various transfusion centers, covering the entire country. All sera were tested for the presence of IgG and IgM antibodies, using ELISAs with various antigenic specificities: (1) heat-denatured coxsackievirus type B1 and B5 virions, (2) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B3, and (3) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B4. Sera positive for IgG antibodies against coxsackieviruses B1/B5, B3, and B4 were detected in 6.7 to 21.6% of the individuals tested in the various regions of Greece. Statistical analysis revealed that the highest prevalence of IgG antibodies against coxsackieviruses B1/B5 was found in blood donors from Crete (p = 0.025), whereas the highest prevalence against coxsackievirus B4 was detected in blood donors from Athens (p = 0.01). IgM antibodies against coxsackievirus B were detected at low percentage, less than 5%, with no significant viral preference for particular geographic regions. The preference of anti-coxsackievirus IgG antibodies for particular geographic regions could be potentially related to the previously reported clustering of cases of insulin-dependent diabetes mellitus and myocarditis in Athens and Crete, respectively.