We report a retrospective analysis carried out on 23,485 subjects submitted to a screening program from 2000 to 2006. Of these subjects, 3,934 had borderline HbA2 values from 3.1 to 3.9%; 410 samples, analyzed previously using PCR methods and sequencing because all of these were partners of a carrier of classical β-thalassemia, were selected for statistical analysis. Of 410 subjects, 94 (22.9%) were positive for a molecular defect in the β-, δ- or α-globin genes. The most prevalent molecular defects were β IVS1 nt 6 (HBB c.92+6T C), co-inheritance of severe β thalassemia and δ mutations, β-promoter mutations and triplication of α genes were detected; α-thalassemia and Hb-variants were also evident. Borderline HbA2 is not a rare event in a population with a high prevalence of β-thalassemia carriers. These data support the necessity to investigate these cases at a molecular level, particularly if the partner is a carrier of β-thalassemia.