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Aims: To investigate the effect of heterozygous β-thalassemia on HbA1c levels in a population without diabetes mellitus (DM). Methods: Using a cross-sectional design, HbA1c levels were compared between two groups of 100 consecutive carriers of β-thalassemia and 100 healthy controls matched for age, gender and BMI, taking into account fasting serum glucose and fructosamine levels. The effect of hemoglobin concentration on HbA1c was also examined. Results: The mean HbA1c level was almost identical in the two groups (33.6 mmol/mol [5.23%] vs. 33.6 mmol/mol [5.22%], p = 0.857). Within the group of β-thalassemia, there was a positive correlation between HbA1c and hemoglobin concentration (r = 0.455, p < 0.001), which was not observed in controls. β-thalassemia carriers without anemia had slightly higher HbA1c levels compared to those with anemia (34.9 mmol/mol [5.35%] vs. 32.5 mmol/mol [5.12%] p < 0.001, absolute difference (2.4 mmol/mol [0.23%]). In multivariable analysis, hemoglobin concentration, BMI and 1st degree family history of T2DM were significant predictors of HbA1c, while β-thalassemia carrier state was non-significant (p = 0.07). Conclusions: In individuals without DM, heterozygous β-thalassemia has a borderline effect on HbA1c levels, while the impact of β-thalassemia trait-associated anemia on HbA1c is of negligible clinical significance. These findings advocate for the clinical use of HbA1c as a diagnostic criterion for diabetes mellitus in this population. © 2019 Elsevier B.V.