Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by continued expression of fetal hemoglobin (HbF) in adulthood. HPFH may be due not only to point mutations or large deletions in different regions of the cluster β globin, but also to variations in several polymorphic sequences in this cluster. The objective of this work was to evaluate effects of polymorphic markers within cluster β globin on HbF expression. For the purpose, we have explored in this first study of Tunisian HPFH four polymorphic regions of β globin cluster in 68 healthy adults (34 subjects with high levels of HbF and 34 with normal HbF levels). Our results showed that the increase of HbF levels is associated with the −158 Gγ C → T polymorphism, the TG18CG2CACG, TC TG9AG TG2CG2 and TG11CG4 configurations in the second intron of Gγ gene and the −540 β (AT)6T9 and (AT)7T8 repeated sequences. Among the 34 subjects with raised levels of HbF, approximately 97% carried one or more of these six markers. This study suggests that there is a significant association between certain polymorphic configurations of the β globin cluster and the increase of HbF levels in healthy individuals.