Objective: To study the possible association between high levels of fetal haemoglobin (HbF) in β-thalassemia intermedia patients and HS-111 and 3`HS1 sequence variations. Materials and Methods: In this study the 3' HS-1 and HS-111 regions of 30 ß-thalassaemia intermedia patients (ß°/ß°) with high levels of HbF 21 ß-thalassemia major patients and 40 normal Iranian individuals were analyzed by single-strand conformation polymorphism (SSCP) and polymerase chain reaction (PCR) sequencing. Results: Two nucleotide variations in 3' HS111 (-21A>G) and 3`HS1 (179C>T) were identified. The most frequent sequence variation was 3' HS111 (-21A) in the intermedia patients and 3`HS111 (-21G) in the major thalassemia patients. In contrast to the 3`HS1 marker both 3'HS111 A and G variants showed a correlation with each studied group. Conclusion: The HS111 marker in conjunction with other parameters could be used as appropriate genetic markers to discriminate β-thalassemia intermedia patients (β°/β°) with high levels of HbF from β-thalassemia major patients.