Abstract The role of the human cytomegalovirus (HCMV) infection in individuals with hemoglobinopathies is unclear. Our objective was to examine the molecular and genotypic characteristics of HCMV in patients with sickle cell disease, beta-thalassemia major, and volunteer blood donors by viral load quantitation, glycoprotein B (gB) genotyping, and phylogenetic analysis. The patients with sickle cell disease demonstrated the highest HCMV DNA prevalence (13.8%), followed by the patients with beta-thalassemia major (7.6%), and the blood donors (3%). The infection was characterized by a low mean viral load (3.8×103 copies/mL), but infections with higher copy numbers were also observed. Genotype gB2 was detected in the majority of cases (90.9%), followed by genotype gB1 (9.1%). No gB3/gB4 genotype was detected. No statistical significance was observed between HCMV DNAemia/gB genotype and hematological alterations or severity of the disease. The high number of sickle cell disease patients with HCMV DNAemia could be due to their partial immune dysfunction (multiple transfusions, spleen dysfunction, hydroxyurea treatment). The extensive HCMV gB2 prevalence in patients with hemoglobinopathies is probably due to HCMV epidemiologic characteristics in the examined region, and can be important during the clinical management of these patients.